Methods of Treating Chronic Lymphocytic Leukemia and Small Lymphocytic Leukemia Using a BTK Inhibitor

ABSTRACT

Therapeutic methods of treating chronic lymphocytic leukemia (CLL) and small lymphocytic leukemia (SLL) are described. In certain embodiments, the invention includes therapeutic methods of treating CLL and SLL using a BTK inhibitor. In certain embodiments, the invention includes therapeutic methods of treating subtypes of CLL and SLL using a BTK inhibitor, including subtypes of CLL in patients sensitive to thrombosis and subtypes of CLL that increase monocytes and NK cells in peripheral blood after treatment with a BTK inhibitor. In certain embodiments, the invention includes therapeutic methods of treating CLL and SLL using a combination of a BTK inhibitor and an anti-CD20 antibody.

FIELD OF THE INVENTION

Therapeutic methods of treating chronic lymphocytic leukemia using aBruton's tyrosine kinase (BTK) inhibitor are disclosed herein.

BACKGROUND OF THE INVENTION

Bruton's Tyrosine Kinase (BTK or Btk) is a TEC family non-receptorprotein kinase expressed in B cells and myeloid cells. The function ofBTK in signaling pathways activated by the engagement of the B cellreceptor (BCR) and FCER1 on mast cells is well established. Functionalmutations in BTK in humans result in a primary immunodeficiency diseasecharacterized by a defect in B cell development with a block betweenpro- and pre-B cell stages. The result is an almost complete absence ofB lymphocytes, causing a pronounced reduction of serum immunoglobulin ofall classes. These findings support a key role for BTK in the regulationof the production of auto-antibodies in autoimmune diseases.

Other diseases with an important role for dysfunctional B cells are Bcell malignancies. The reported role for BTK in the regulation ofproliferation and apoptosis of B cells indicates the potential for BTKinhibitors in the treatment of B cell lymphomas. BTK inhibitors havethus been developed as potential therapies, as described in O. J. D'Cruzand F. M. Uckun, OncoTargets and Therapy 2013, 6, 161-176.

B cell chronic lymphocytic leukemia (CLL) is one of the most prevalent Bcell malignancies in adults. CLL is characterized by an expansion ofmonoclonal mature B cells. CLL patients who relapsed after standardtreatments generally experience poor outcomes. Although survival hasbeen improved by the addition of immunotherapies such as rituximab tostandard chemotherapies such as fludarabine and cyclophosphamide, asdescribed in M. Hallek, et al., Lancet, 2010, 76, 1164-74, many standardtreatments are associated with toxicities and immunosuppression. Thereis therefore a significant need to identify less toxic and highlyefficacious treatments for CLL. Small lymphocytic leukemia (SLL) isclosely related to CLL, and differs only in that a lower level ofmonoclonal lymphocytes is observed in blood than in CLL, along with anenlarged spleen or lymph nodes. There is also a significant need toidentify less toxic and highly efficacious treatments for SLL.

CLL (and SLL) cells rapidly accumulate and are resistant to apoptosis invivo, but are known to die rapidly in vitro. M. Buchner, et al., Blood2010, 115, 4497-506. One cause of this effect is from nonmalignantaccessory cells in the tumor microenvironment, such as stromal cellcontact mediated cell survival. Stromal cells in the bone marrow andlymph nodes are known to have an antiapoptotic and protective effect onCLL cells, protecting them from both chemotherapeutic and spontaneousapoptosis. R. E. Mudry, et al., Blood 2000, 96, 1926-32. The chemokineSDF1α (CXCL12) directs homing of CLL cells towards protective niches. M.Burger, et al., Blood 2005, 106, 1824-30. Existing drugs that target theBCR pathway in B cell malignancies can lead to some lymphocytosis, i.e.lymphocyte egress from nodal compartments, through disruption ofCXCR4-SDF1α signaling and other adhesion factors in bone marrow and theresulting mobilization of cells. However, existing therapies may noteradicate residual malignant B cell populations in the microenvironmentof the bone marrow and lymph nodes, where protective stromal cellsprevent apoptosis. There is thus an urgent need for treatments thatreduce or overcome the protective effect of the microenvironment on CLLcells to enable superior clinical responses in patients.

SUMMARY OF THE INVENTION

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, wherein the BTK inhibitor is administered once daily ata dose selected from the group consisting of 100 mg, 175 mg, 250 mg, and400 mg.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, wherein the BTK inhibitor is administered twice dailyat a dose of 100 mg.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, wherein the CLL increases monocytes and NK cells inperipheral blood after treatment with Formula (II) for a period selectedfrom the group consisting of about 14 days, about 28 days, or about 56days.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, wherein the CLL is selected from the group consistingof IgVH mutation negative CLL, ZAP-70 positive CLL, ZAP-70 methylated atCpG3 CLL, CD38 positive CLL, CLL with a 17p13.1 (17p) deletion, CLL witha 11q22.3 (11q) deletion, CLL in a human sensitive to platelet-mediatedthrombosis, CLL in a human presently suffering from platelet-mediatedthrombosis, CLL in a human previously suffering from platelet-mediatedthrombosis, or combinations thereof.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, further comprising the step of administering atherapeutically effective dose of an anti-CD20 antibody selected fromthe group consisting of rituximab, obinutuzumab, ofatumumab, veltuzumab,tositumomab, ibritumomab, and fragments, derivatives, conjugates,variants, radioisotope-labeled complexes, and biosimilars thereof.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, further comprising the step of administering atherapeutically effective dose of an anticoagulant or antiplateletactive pharmaceutical ingredient.

In an embodiment, the invention includes a method of treating CLL and/orSLL, comprising the step of orally administering, to a human in needthereof, a Bruton's tyrosine kinase (BTK) inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, further comprising the step of administering atherapeutically effective dose of an anticoagulant or antiplateletactive pharmaceutical ingredient, wherein the anticoagulant orantiplatelet active pharmaceutical ingredient is selected from the groupconsisting of acenocoumarol, anagrelide, anagrelide hydrochloride,abciximab, aloxiprin, antithrombin, apixaban, argatroban, aspirin,aspirin with extended-release dipyridamole, beraprost, betrixaban,bivalirudin, carbasalate calcium, cilostazol, clopidogrel, clopidogrelbisulfate, cloricromen, dabigatran etexilate, darexaban, dalteparin,dalteparin sodium, defibrotide, dicumarol, diphenadione, dipyridamole,ditazole, desirudin, edoxaban, enoxaparin, enoxaparin sodium,eptifibatide, fondaparinux, fondaparinux sodium, heparin, heparinsodium, heparin calcium, idraparinux, idraparinux sodium, iloprost,indobufen, lepirudin, low molecular weight heparin, melagatran,nadroparin, otamixaban, parnaparin, phenindione, phenprocoumon,prasugrel, picotamide, prostacyclin, ramatroban, reviparin, rivaroxaban,sulodexide, terutroban, terutroban sodium, ticagrelor, ticlopidine,ticlopidine hydrochloride, tinzaparin, tinzaparin sodium, tirofiban,tirofiban hydrochloride, treprostinil, treprostinil sodium, triflusal,vorapaxar, warfarin, warfarin sodium, ximelagatran, salts thereof,solvates thereof, hydrates thereof, and combinations thereof.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis, wherein the BTK inhibitoris administered once daily at a dose selected from the group consistingof 100 mg, 175 mg, 250 mg, and 400 mg.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis, wherein the BTK inhibitoris administered twice daily at a dose of 100 mg.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis, wherein the hematologicalmalignancy increases monocytes and NK cells in peripheral blood aftertreatment with Formula (II) for a period selected from the groupconsisting of about 14 days, about 28 days, or about 56 days.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy isnon-Hodgkin's lymphoma (NHL), wherein the NHL is selected from the groupconsisting of indolent NHL and aggressive NHL.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is diffuselarge B cell lymphoma (DLBCL), wherein the DLBCL is selected from thegroup consisting of activated B-cell like diffuse large B-cell lymphoma(DLBCL-ABC) and germinal center B-cell like diffuse large B-celllymphoma (DLBCL-GCB).

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is mantle celllymphoma (MCL), wherein the MCL is selected from the group consisting ofmantle zone MCL, nodular MCL, diffuse MCL, and blastoid MCL.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is B cellacute lymphoblastic leukemia (B-ALL), wherein the B-ALL is selected fromthe group consisting of early pre-B cell B-ALL, pre-B cell B-ALL, andmature B cell B-ALL.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is Burkitt'slymphoma, wherein the Burkitt's lymphoma is selected from the groupconsisting of sporadic Burkitt's lymphoma, endemic Burkitt's lymphoma,and human immunodeficiency virus-associated Burkitt's lymphoma.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is multiplemyeloma, wherein the multiple myeloma is selected from the groupconsisting of hyperdiploid multiple myeloma and non-hyperdiploidmultiple myeloma.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy ismyelofibrosis, wherein the myelofibrosis is selected from the groupconsisting of primary myelofibrosis, myelofibrosis secondary topolycythemia vera, and myelofibrosis secondary to essentialthrombocythaemia.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis, further comprising thestep of administering a therapeutically effective dose of an anti-CD20antibody selected from the group consisting of rituximab, obinutuzumab,ofatumumab, veltuzumab, tositumomab, ibritumomab, and fragments,derivatives, conjugates, variants, radioisotope-labeled complexes, andbiosimilars thereof.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis, further comprising thestep of administering a therapeutically effective dose of ananticoagulant or antiplatelet active pharmaceutical ingredient.

In an embodiment, the invention includes a method of treating ahematological malignancy in a human comprising the step of administeringa therapeutically effective dose of a BTK inhibitor, wherein the BTKinhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof, and wherein the hematological malignancy is selectedfrom the group consisting of non-Hodgkin's lymphoma (NHL), diffuse largeB cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, or myelofibrosis, further comprising thestep of administering a therapeutically effective dose of ananticoagulant or antiplatelet active pharmaceutical ingredient, whereinthe anticoagulant or antiplatelet active pharmaceutical ingredient isselected from the group consisting of acenocoumarol, anagrelide,anagrelide hydrochloride, abciximab, aloxiprin, antithrombin, apixaban,argatroban, aspirin, aspirin with extended-release dipyridamole,beraprost, betrixaban, bivalirudin, carbasalate calcium, cilostazol,clopidogrel, clopidogrel bisulfate, cloricromen, dabigatran etexilate,darexaban, dalteparin, dalteparin sodium, defibrotide, dicumarol,diphenadione, dipyridamole, ditazole, desirudin, edoxaban, enoxaparin,enoxaparin sodium, eptifibatide, fondaparinux, fondaparinux sodium,heparin, heparin sodium, heparin calcium, idraparinux, idraparinuxsodium, iloprost, indobufen, lepirudin, low molecular weight heparin,melagatran, nadroparin, otamixaban, parnaparin, phenindione,phenprocoumon, prasugrel, picotamide, prostacyclin, ramatroban,reviparin, rivaroxaban, sulodexide, terutroban, terutroban sodium,ticagrelor, ticlopidine, ticlopidine hydrochloride, tinzaparin,tinzaparin sodium, tirofiban, tirofiban hydrochloride, treprostinil,treprostinil sodium, triflusal, vorapaxar, warfarin, warfarin sodium,ximelagatran, salts thereof, solvates thereof, hydrates thereof, andcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1 illustrates in vivo potency of Formula (II) (labeled “BTKinhibitor”) and ibrutinib. Mice were gavaged at increasing drugconcentration and sacrificed at one time point (3 h post-dose). BCR isstimulated with IgM and the expression of activation markers CD69 andCD86 are monitored by flow cytometry to determine EC₅₀'s. The resultsshow that Formula (II) is more potent at inhibiting expression ofactivation makers than ibrutinib.

FIG. 2 illustrates the results of the clinical study of Formula (II)(labeled “BTK inhibitor”) in CLL, which are shown in comparison to theresults reported for ibrutinib in FIG. 1A of J. C. Byrd, et al., N.Engl. J. Med. 2013, 369, 32-42. The results show that the BTK inhibitorof Formula (II) causes a much smaller relative increase and much fasterdecrease in absolute lymphocyte count (ALC) relative to the BTKinhibitor ibrutinib. The sum of the product of greatest diameters (SPD)also decreases more rapidly during treatment with the BTK inhibitor thanwith the BTK inhibitor ibrutinib.

FIG. 3 shows overall response data shown by SPD of enlarged lymph nodesin CLL patients as a function of dose of the BTK inhibitor of Formula(II).

FIG. 4 shows a comparison of progression-free survival (PFS) in CLLpatients treated with the BTK inhibitor ibrutinib or the BTK inhibitorof Formula (II). The ibrutinib data is taken from J. C. Byrd, et al., N.Engl. J. Med. 2013, 369, 32-42. CLL patients treated with Formula (II)for at least 8 days are included.

FIG. 5 shows a comparison of number of patients at risk in CLL patientstreated with the BTK inhibitor ibrutinib or the BTK inhibitor of Formula(II). CLL patients treated with Formula (II) for at least 8 days areincluded.

FIG. 6 shows a comparison of progression-free survival (PFS) in CLLpatients exhibiting the 17p deletion and treated with the BTK inhibitoribrutinib or the BTK inhibitor of Formula (II). The ibrutinib data istaken from J. C. Byrd, et al., N. Engl. J. Med. 2013, 369, 32-42.

FIG. 7 shows a comparison of number of patients at risk in CLL patientsexhibiting the 17p deletion and treated with the BTK inhibitor ibrutinibor the BTK inhibitor of Formula (II). The ibrutinib data is taken fromJ. C. Byrd, et al., N. Engl. J. Med. 2013, 369, 32-42. CLL patientstreated with Formula (II) for at least 8 days are included.

FIG. 8 shows improved BTK target occupancy of Formula (II) at lowerdosage versus ibrutinib in relapsed/refractory CLL patients.

FIG. 9 shows the % change in myeloid-derived suppressor cell (MDSC)(monocytic) level over 28 days versus % ALC change at Cycle 1, day 28(C1D28) with trendlines.

FIG. 10 shows the % change in MDSC (monocytic) level over 28 days versus% ALC change at Cycle 2, day 28 (C2D28) with trendlines.

FIG. 11 shows the % change in natural killer (NK) cell level over 28days versus % ALC change at Cycle 1, day 28 (C2D28) with trendlines.

FIG. 12 shows the % change in NK cell level over 28 days versus % ALCchange at Cycle 2, day 28 (C2D28) with trendlines.

FIG. 13 compares the % change in MDSC (monocytic) level and % change inNK cell level over 28 days versus % ALC change with the % change inlevel of CD4⁺ T cells, CD8⁺ T cells, CD4⁺/CD8⁺ T cell ratio, NK-T cells,PD-1⁺ CD4⁺ T cells, and PD-1⁺ CD8⁺ T cells, also versus % ALC change, atCycle 1 day 28 (C1D28). Trendlines are shown for % change in MDSC(monocytic) level and % change in NK cell level.

FIG. 14 compares the % change in MDSC (monocytic) level and % change inNK cell level over 28 days versus % ALC change with the % change inlevel of CD4⁺ T cells, CD8⁺ T cells, CD4⁺/CD8⁺ T cell ratio, NK-T cells,PD-1⁺ CD4⁺ T cells, and PD-1⁺ CD8⁺ T cells, also versus % ALC change, atCycle 2 day 28 (C2D28). Trendlines are shown for % change in MDSC(monocytic) level and % change in NK cell level.

FIG. 15 illustrates representative photomicrographs and comparison ofmaximal thrombus size in laser injured arterioles of VWF HA1 mutant miceinfused with human platelets in the absence or presence of various BTKinhibitors. Representative photomicrographs are given as a comparison ofmaximal thrombus size in laser-injured arterioles (1 μM concentrationsshown).

FIG. 16 illustrates a quantitative comparison obtained by in vivoanalysis of early thrombus dynamics in a humanized mouse laser injurymodel using three BTK inhibitors at a concentration of 1 μM.

FIG. 17 illustrates the effect of the tested BTK inhibitors on thrombusformation. The conditions used were N=4, 3 mice per drug; anti-clottingagents <2000 μM². In studies with ibrutinib, 48% MCL bleeding eventswere observed with 560 mg QD and 63% CLL bleeding events were observedwith 420 mg QD, where bleeding event is defined as subdural hematoma,ecchymoses, GI bleeding, or hematuria.

FIG. 18 illustrates the effect of the concentration of the tested BTKinhibitors on thrombus formation.

FIG. 19 illustrates the results of platelet collagen receptorglycoprotein VI (GPVI) platelet aggregation studies of Formula (II)(IC₅₀=1.15 μM) and ibrutinib (IC₅₀=0.13 μM).

FIG. 20 illustrates the results of GPVI platelet aggregation studies ofFormula (II) and ibrutinib.

FIG. 21 shows in vitro analysis of antibody-dependent NK cell-mediatedINF-γ release with BTK inhibitors. To evaluate NK cell function,purified NK cells were isolated from healthy peripheral bloodmononuclear cells and cultured with 0.1 or 1 μM of ibrutinib or 1 μM ofFormula (II) for 4 hours together with rituximab-coated (10 μg/mL)lymphoma cells, DHL4, or trastuzumab-coated (10 μg/mL) HER2+ breastcancer cells, HER18, and supernatant was harvested and analyzed byenzyme-linked immunosorbent assay for interferon-γ (IFN-γ). All in vitroexperiments were performed in triplicate. Labels are defined as follows:*p=0.018, **p=0.002, ***p=0.001.

FIG. 22 shows in vitro analysis of antibody-dependent NK cell-mediateddegranulation with BTK inhibitors. To evaluate NK cell function,purified NK cells were isolated from healthy peripheral bloodmononuclear cells and cultured with 0.1 or 1 μM of ibrutinib or 1 μM ofFormula (II) for 4 hours together with rituximab-coated (10 μg/mL)lymphoma cells, DHL4, or trastuzumab-coated (10 μg/mL) HER2+ breastcancer cells, HER18, and NK cells isolated and analyzed fordegranulation by flow cytometry for CD107a mobilization. All in vitroexperiments were performed in triplicate. Labels are defined as follows:*p=0.01, **p=0.002, ***p=0.003, ****p=0.0005.

FIG. 23 shows that ibrutinib antagonizes antibody-dependent NKcell-mediated cytotoxicity in primary CLL cells. NK cell cytotoxicity aspercent lysis of tumor cells was analyzed in chromium release assayswith purified NK cells incubated with chromium-labeled Raji for 4 hoursat variable rituximab concentrations at a constant effector:target ratioof 25:1 and ibrutinib (1 μM), Formula (II) (1 μM), or other ITK sparingBTK inhibitors CGI-1746, inhibA (1 μM) and BGB-3111 (1 μM). All in vitroexperiments were performed in triplicate. Labels are defined as follows:*p=0.001.

FIG. 24 shows a summary of the results given in FIG. 24 at the highestconcentration of rituximab (“Ab”) (10 μg/mL).

FIG. 25 shows that ibrutinib antagonizes antibody-dependent NKcell-mediated cytotoxicity, as in FIG. 23, using the Raji cell line.

FIG. 26 shows the effects of BTK inhibition on generalized NK cellmediated cytotoxicity.

FIG. 27 shows that Formula (II) has no adverse effect on T helper 17(Th17) cells, which are a subset of T helper cells that produceinterleukin 17 (IL17), while ibrutinib strongly inhibits Th17 cells.

FIG. 28 shows that Formula (II) has no effect on regulatory T cell(Treg) development, while ibrutinib strongly increases Treg development.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO:1 is the heavy chain amino acid sequence of the anti-CD20monoclonal antibody rituximab.

SEQ ID NO:2 is the light chain amino acid sequence of the anti-CD20monoclonal antibody rituximab.

SEQ ID NO:3 is the heavy chain amino acid sequence of the anti-CD20monoclonal antibody obinutuzumab.

SEQ ID NO:4 is the light chain amino acid sequence of the anti-CD20monoclonal antibody obinutuzumab.

SEQ ID NO:5 is the variable heavy chain amino acid sequence of theanti-CD20 monoclonal antibody ofatumumab.

SEQ ID NO:6 is the variable light chain amino acid sequence of theanti-CD20 monoclonal antibody ofatumumab.

SEQ ID NO:7 is the Fab fragment heavy chain amino acid sequence of theanti-CD20 monoclonal antibody ofatumumab.

SEQ ID NO:8 is the Fab fragment light chain amino acid sequence of theanti-CD20 monoclonal antibody ofatumumab.

SEQ ID NO:9 is the heavy chain amino acid sequence of the anti-CD20monoclonal antibody veltuzumab.

SEQ ID NO:10 is the light chain amino acid sequence of the anti-CD20monoclonal antibody veltuzumab.

SEQ ID NO:11 is the heavy chain amino acid sequence of the anti-CD20monoclonal antibody tositumomab.

SEQ ID NO:12 is the light chain amino acid sequence of the anti-CD20monoclonal antibody tositumomab.

SEQ ID NO:13 is the heavy chain amino acid sequence of the anti-CD20monoclonal antibody ibritumomab.

SEQ ID NO:14 is the light chain amino acid sequence of the anti-CD20monoclonal antibody ibritumomab.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the invention are shown and describedherein, such embodiments are provided by way of example only and are notintended to otherwise limit the scope of the invention. Variousalternatives to the described embodiments of the invention may beemployed in practicing the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs.

The terms “co-administration” and “administered in combination with” asused herein, encompass administration of two or more activepharmaceutical ingredients to a subject so that both agents and/or theirmetabolites are present in the subject at the same time.Co-administration includes simultaneous administration in separatecompositions, administration at different times in separatecompositions, or administration in a composition in which two or moreagents are present.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “IC₅₀” refers to the half maximal inhibitory concentration,i.e. inhibition of 50% of the desired activity. The term “EC₅₀” refersto the drug concentration at which one-half the maximum response isachieved.

The term “effective amount” or “therapeutically effective amount” refersto that amount of an active pharmaceutical ingredient or combination ofactive pharmaceutical ingredients as described herein that is sufficientto effect the intended application including, but not limited to,disease treatment. A therapeutically effective amount may vary dependingupon the intended application (in vitro or in vivo), or the subject anddisease condition being treated (e.g., the weight, age and gender of thesubject), the severity of the disease condition, the manner ofadministration, and other factors which can readily be determined by oneof ordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, (e.g., the reduction ofplatelet adhesion and/or cell migration). The specific dose will varydepending on the particular compounds chosen, the dosing regimen to befollowed, whether the compound is administered in combination with othercompounds, timing of administration, the tissue to which it isadministered, and the physical delivery system in which the compound iscarried.

A “therapeutic effect” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

The terms “QD,” “qd,” or “q.d.” means quaque die, once a day, or oncedaily. The terms “BID,” “bid,” or “b.i.d.” mean bis in die, twice a day,or twice daily. The terms “TID,” “tid,” or “t.i.d.” mean ter in die,three times a day, or three times daily. The terms “QID,” “qid,” or“q.i.d.” mean quater in die, four times a day, or four times daily.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions known in the art.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid and phosphoric acid. Organic acids from whichsalts can be derived include, for example, acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid and salicylic acid. Pharmaceutically acceptablebase addition salts can be formed with inorganic and organic bases.Inorganic bases from which salts can be derived include, for example,sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese and aluminum. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins. Specific examples includeisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. In selected embodiments, thepharmaceutically acceptable base addition salt is chosen from ammonium,potassium, sodium, calcium, and magnesium salts. The term “cocrystal”refers to a molecular complex derived from a number of cocrystal formersknown in the art. Unlike a salt, a cocrystal typically does not involveproton transfer between the cocrystal and the drug, and instead involvesintermolecular interactions, such as hydrogen bonding, aromatic ringstacking, or dispersive forces, between the cocrystal former and thedrug in the crystal structure.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” is intended to include any and all solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic, andabsorption delaying agents. The use of such media and agents for activepharmaceutical ingredients is well known in the art. Except insofar asany conventional media or agent is incompatible with the activepharmaceutical ingredient, its use in the therapeutic compositions ofthe invention is contemplated. Supplementary active ingredients can alsobe incorporated into the described compositions.

“Prodrug” is intended to describe a substance that may be convertedunder physiological conditions or by solvolysis to a biologically activepharmaceutical ingredient described herein. Thus, the term “prodrug”refers to a precursor of a biologically active compound that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject, but is converted in vivo to an active pharmaceuticalingredient, for example, by hydrolysis. The prodrug compound oftenoffers the advantages of solubility, tissue compatibility or delayedrelease in a mammalian organism (see, e.g., H. Bundgaard, Design ofProdrugs, Elsevier, Amsterdam (1985)). The term “prodrug” is alsointended to include any covalently bonded carriers, which release theactive pharmaceutical ingredient in vivo when administered to a subject.Prodrugs of an active pharmaceutical ingredient, as described herein,may be prepared by modifying functional groups present in the activepharmaceutical ingredient in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the activepharmaceutical ingredient. Prodrugs include, for example, compoundswherein a hydroxy, amino or mercapto group is bonded to any group that,when the prodrug of the active pharmaceutical ingredient is administeredto a mammalian subject, cleaves to form a free hydroxy, free amino orfree mercapto group, respectively. Examples of prodrugs include, but arenot limited to, acetates, formates and benzoate derivatives of analcohol, various ester derivatives of a carboxylic acid, or acetamide,formamide and benzamide derivatives of an amine functional group in theactive pharmaceutical ingredient.

When ranges are used herein to describe, for example, physical orchemical properties such as molecular weight or chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. Use of the term “about” whenreferring to a number or a numerical range means that the number ornumerical range referred to is an approximation within experimentalvariability (or within statistical experimental error), and thus thenumber or numerical range may vary from, for example, between 1% and 15%of the stated number or numerical range. The term “comprising” (andrelated terms such as “comprise” or “comprises” or “having” or“including”) includes those embodiments such as, for example, anembodiment of any composition of matter, method or process that “consistof” or “consist essentially of” the described features.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to ten carbon atoms (e.g., C₁-C₁₀ alkyl).Whenever it appears herein, a numerical range such as “1 to 10” refersto each integer in the given range—e.g., “1 to 10 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 10 carbon atoms, although thedefinition is also intended to cover the occurrence of the term “alkyl”where no numerical range is specifically designated. Typical alkylgroups include, but are in no way limited to, methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, sec-butyl isobutyl, tertiary butyl,pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl and decyl. Thealkyl moiety may be attached to the rest of the molecule by a singlebond, such as for example, methyl (Me), ethyl (Et), n-propyl (Pr),1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl) and 3-methylhexyl. Unless stated otherwise specifically in thespecification, an alkyl group is optionally substituted by one or moreof substituents which are independently alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂ whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkylaryl” refers to an -(alkyl)aryl radical where aryl and alkyl areas disclosed herein and which are optionally substituted by one or moreof the substituents described as suitable substituents for aryl andalkyl respectively.

“Alkylhetaryl” refers to an -(alkyl)hetaryl radical where hetaryl andalkyl are as disclosed herein and which are optionally substituted byone or more of the substituents described as suitable substituents foraryl and alkyl respectively.

“Alkylheterocycloalkyl” refers to an -(alkyl) heterocycyl radical wherealkyl and heterocycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heterocycloalkyl and alkyl respectively.

An “alkene” moiety refers to a group consisting of at least two carbonatoms and at least one carbon-carbon double bond, and an “alkyne” moietyrefers to a group consisting of at least two carbon atoms and at leastone carbon-carbon triple bond. The alkyl moiety, whether saturated orunsaturated, may be branched, straight chain, or cyclic.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one double bond, and having from two to ten carbon atoms (i.e.,C₂-C₁₀ alkenyl). Whenever it appears herein, a numerical range such as“2 to 10” refers to each integer in the given range—e.g., “2 to 10carbon atoms” means that the alkenyl group may consist of 2 carbonatoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. Thealkenyl moiety may be attached to the rest of the molecule by a singlebond, such as for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e.,allyl), but-1-enyl, pent-1-enyl and penta-1,4-dienyl. Unless statedotherwise specifically in the specification, an alkenyl group isoptionally substituted by one or more substituents which areindependently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkenyl-cycloalkyl” refers to an -(alkenyl)cycloalkyl radical wherealkenyl and cycloalkyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for alkenyl and cycloalkyl respectively.

“Alkynyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one triple bond, having from two to ten carbon atoms (i.e. C₂-C₁₀alkynyl). Whenever it appears herein, a numerical range such as “2 to10” refers to each integer in the given range—e.g., “2 to 10 carbonatoms” means that the alkynyl group may consist of 2 carbon atoms, 3carbon atoms, etc., up to and including 10 carbon atoms. The alkynyl maybe attached to the rest of the molecule by a single bond, for example,ethynyl, propynyl, butynyl, pentynyl and hexynyl. Unless statedotherwise specifically in the specification, an alkynyl group isoptionally substituted by one or more substituents which independentlyare: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Alkynyl-cycloalkyl” refers to an -(alkynyl)cycloalkyl radical wherealkynyl and cycloalkyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for alkynyl and cycloalkyl respectively.

“Carboxaldehyde” refers to a —(C═O)H radical.

“Carboxyl” refers to a —(C═O)OH radical.

“Cyano” refers to a —CN radical.

“Cycloalkyl” refers to a monocyclic or polycyclic radical that containsonly carbon and hydrogen, and may be saturated, or partiallyunsaturated. Cycloalkyl groups include groups having from 3 to 10 ringatoms (i.e. C₂-C₁₀ cycloalkyl). Whenever it appears herein, a numericalrange such as “3 to 10” refers to each integer in the given range—e.g.,“3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3carbon atoms, etc., up to and including 10 carbon atoms. Illustrativeexamples of cycloalkyl groups include, but are not limited to thefollowing moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloseptyl, cyclooctyl, cyclononyl,cyclodecyl, norbornyl, and the like. Unless stated otherwisespecifically in the specification, a cycloalkyl group is optionallysubstituted by one or more substituents which independently are: alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Cycloalkyl-alkenyl” refers to a -(cycloalkyl)alkenyl radical wherecycloalkyl and alkenyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for cycloalkyl and alkenyl, respectively.

“Cycloalkyl-heterocycloalkyl” refers to a -(cycloalkyl)heterocycloalkylradical where cycloalkyl and heterocycloalkyl are as disclosed hereinand which are optionally substituted by one or more of the substituentsdescribed as suitable substituents for cycloalkyl and heterocycloalkyl,respectively.

“Cycloalkyl-heteroaryl” refers to a -(cycloalkyl)heteroaryl radicalwhere cycloalkyl and heteroaryl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for cycloalkyl and heteroaryl, respectively.

The term “alkoxy” refers to the group —O-alkyl, including from 1 to 8carbon atoms of a straight, branched, cyclic configuration andcombinations thereof attached to the parent structure through an oxygen.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, cyclopropyloxy and cyclohexyloxy. “Lower alkoxy” refers toalkoxy groups containing one to six carbons.

The term “substituted alkoxy” refers to alkoxy wherein the alkylconstituent is substituted (i.e., —O-(substituted alkyl)). Unless statedotherwise specifically in the specification, the alkyl moiety of analkoxy group is optionally substituted by one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

The term “alkoxycarbonyl” refers to a group of the formula(alkoxy)(C═O)— attached through the carbonyl carbon wherein the alkoxygroup has the indicated number of carbon atoms. Thus a C₁-C₆alkoxycarbonyl group is an alkoxy group having from 1 to 6 carbon atomsattached through its oxygen to a carbonyl linker. “Lower alkoxycarbonyl”refers to an alkoxycarbonyl group wherein the alkoxy group is a loweralkoxy group.

The term “substituted alkoxycarbonyl” refers to the group (substitutedalkyl)-O—C(O)— wherein the group is attached to the parent structurethrough the carbonyl functionality. Unless stated otherwise specificallyin the specification, the alkyl moiety of an alkoxycarbonyl group isoptionally substituted by one or more substituents which independentlyare: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Acyl” refers to the groups (alkyl)-C(O)—, (aryl)-C(O)—,(heteroaryl)-C(O)—, (heteroalkyl)-C(O)— and (heterocycloalkyl)-C(O)—,wherein the group is attached to the parent structure through thecarbonyl functionality. If the R radical is heteroaryl orheterocycloalkyl, the hetero ring or chain atoms contribute to the totalnumber of chain or ring atoms. Unless stated otherwise specifically inthe specification, the alkyl, aryl or heteroaryl moiety of the acylgroup is optionally substituted by one or more substituents which areindependently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Acyloxy” refers to a R(C═O)O— radical wherein R is alkyl, aryl,heteroaryl, heteroalkyl or heterocycloalkyl, which are as describedherein. If the R radical is heteroaryl or heterocycloalkyl, the heteroring or chain atoms contribute to the total number of chain or ringatoms. Unless stated otherwise specifically in the specification, the Rof an acyloxy group is optionally substituted by one or moresubstituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Amino” or “amine” refers to a —N(R^(a))₂ radical group, where eachR^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless statedotherwise specifically in the specification. When a —N(R^(a))₂ group hastwo R^(a) substituents other than hydrogen, they can be combined withthe nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example,—N(R^(a))₂ is intended to include, but is not limited to, 1-pyrrolidinyland 4-morpholinyl. Unless stated otherwise specifically in thespecification, an amino group is optionally substituted by one or moresubstituents which independently are: alkyl, heteroalkyl, alkenyl,alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

The term “substituted amino” also refers to N-oxides of the groups—NHR^(d), and NR^(d)R^(d) each as described above. N-oxides can beprepared by treatment of the corresponding amino group with, forexample, hydrogen peroxide or m-chloroperoxybenzoic acid.

“Amide” or “amido” refers to a chemical moiety with formula —C(O)N(R)₂or —NHC(O)R, where R is selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon), each of which moiety mayitself be optionally substituted. The R₂ of —N(R)₂ of the amide mayoptionally be taken together with the nitrogen to which it is attachedto form a 4-, 5-, 6- or 7-membered ring. Unless stated otherwisespecifically in the specification, an amido group is optionallysubstituted independently by one or more of the substituents asdescribed herein for alkyl, cycloalkyl, aryl, heteroaryl, orheterocycloalkyl. An amide may be an amino acid or a peptide moleculeattached to a compound of Formula (I), thereby forming a prodrug. Theprocedures and specific groups to make such amides are known to those ofskill in the art and can readily be found in seminal sources such as T.H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,3^(rd) Ed., John Wiley & Sons, New York (1999).

“Aromatic” or “aryl” or “Ar” refers to an aromatic radical with six toten ring atoms (e.g., C₆-C₁₀ aromatic or C₆-C₁₀ aryl) which has at leastone ring having a conjugated pi electron system which is carbocyclic(e.g., phenyl, fluorenyl, and naphthyl). Bivalent radicals formed fromsubstituted benzene derivatives and having the free valences at ringatoms are named as substituted phenylene radicals. Bivalent radicalsderived from univalent polycyclic hydrocarbon radicals whose names endin “-yl” by removal of one hydrogen atom from the carbon atom with thefree valence are named by adding “-idene” to the name of thecorresponding univalent radical, e.g., a naphthyl group with two pointsof attachment is termed naphthylidene. Whenever it appears herein, anumerical range such as “6 to 10” refers to each integer in the givenrange; e.g., “6 to 10 ring atoms” means that the aryl group may consistof 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms.The term includes monocyclic or fused-ring polycyclic (i.e., rings whichshare adjacent pairs of ring atoms) groups. Unless stated otherwisespecifically in the specification, an aryl moiety is optionallysubstituted by one or more substituents which are independently alkyl,heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, —OR^(a),—SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Aralkyl” or “arylalkyl” refers to an (aryl)alkyl-radical where aryl andalkyl are as disclosed herein and which are optionally substituted byone or more of the substituents described as suitable substituents foraryl and alkyl respectively.

“Ester” refers to a chemical radical of formula —COOR, where R isselected from the group consisting of alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heteroalicyclic (bondedthrough a ring carbon). The procedures and specific groups to makeesters are known to those of skill in the art and can readily be foundin seminal sources such as T. H. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, New York(1999). Unless stated otherwise specifically in the specification, anester group is optionally substituted by one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Fluoroalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more fluoro radicals, as defined above, forexample, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of thefluoroalkyl radical may be optionally substituted as defined above foran alkyl group.

“Halo,” “halide,” or, alternatively, “halogen” is intended to meanfluoro, chloro, bromo or iodo. The terms “haloalkyl,” “haloalkenyl,”“haloalkynyl” and “haloalkoxy” include alkyl, alkenyl, alkynyl andalkoxy structures that are substituted with one or more halo groups orwith combinations thereof. For example, the terms “fluoroalkyl” and“fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, inwhich the halo is fluorine.

“Heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” include optionallysubstituted alkyl, alkenyl and alkynyl radicals and which have one ormore skeletal chain atoms selected from an atom other than carbon, e.g.,oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Anumerical range may be given—e.g., C₁-C₄ heteroalkyl which refers to thechain length in total, which in this example is 4 atoms long. Aheteroalkyl group may be substituted with one or more substituents whichindependently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂,—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))C(O)N(R^(a))₂, N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or 2), or PO₃(R^(a))₂, whereeach R^(a) is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Heteroalkylaryl” refers to an -(heteroalkyl)aryl radical whereheteroalkyl and aryl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for heteroalkyl and aryl, respectively.

“Heteroalkylheteroaryl” refers to an -(heteroalkyl)heteroaryl radicalwhere heteroalkyl and heteroaryl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heteroalkyl and heteroaryl, respectively.

“Heteroalkylheterocycloalkyl” refers to an-(heteroalkyl)heterocycloalkyl radical where heteroalkyl andheterocycloalkyl are as disclosed herein and which are optionallysubstituted by one or more of the substituents described as suitablesubstituents for heteroalkyl and heterocycloalkyl, respectively.

“Heteroalkylcycloalkyl” refers to an -(heteroalkyl)cycloalkyl radicalwhere heteroalkyl and cycloalkyl are as disclosed herein and which areoptionally substituted by one or more of the substituents described assuitable substituents for heteroalkyl and cycloalkyl, respectively.

“Heteroaryl” or “heteroaromatic” or “HetAr” refers to a 5- to18-membered aromatic radical (e.g., C₅-C₁₃ heteroaryl) that includes oneor more ring heteroatoms selected from nitrogen, oxygen and sulfur, andwhich may be a monocyclic, bicyclic, tricyclic or tetracyclic ringsystem. Whenever it appears herein, a numerical range such as “5 to 18”refers to each integer in the given range—e.g., “5 to 18 ring atoms”means that the heteroaryl group may consist of 5 ring atoms, 6 ringatoms, etc., up to and including 18 ring atoms. Bivalent radicalsderived from univalent heteroaryl radicals whose names end in “-yl” byremoval of one hydrogen atom from the atom with the free valence arenamed by adding “-idene” to the name of the corresponding univalentradical—e.g., a pyridyl group with two points of attachment is apyridylidene. A N-containing “heteroaromatic” or “heteroaryl” moietyrefers to an aromatic group in which at least one of the skeletal atomsof the ring is a nitrogen atom. The polycyclic heteroaryl group may befused or non-fused. The heteroatom(s) in the heteroaryl radical areoptionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heteroaryl may be attached to the rest ofthe molecule through any atom of the ring(s). Examples of heteroarylsinclude, but are not limited to, azepinyl, acridinyl, benzimidazolyl,benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl,benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl,benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl,benzothiazolyl, benzothienyl(benzothiophenyl),benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl,pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl,pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl,quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e.thienyl). Unless stated otherwise specifically in the specification, aheteroaryl moiety is optionally substituted by one or more substituentswhich are independently: alkyl, heteroalkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or2), or PO₃(R^(a))₂, where each R^(a) is independently hydrogen, alkyl,fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

Substituted heteroaryl also includes ring systems substituted with oneor more oxide (—O—) substituents, such as, for example, pyridinylN-oxides.

“Heteroarylalkyl” refers to a moiety having an aryl moiety, as describedherein, connected to an alkylene moiety, as described herein, whereinthe connection to the remainder of the molecule is through the alkylenegroup.

“Heterocycloalkyl” refers to a stable 3- to 18-membered non-aromaticring radical that comprises two to twelve carbon atoms and from one tosix heteroatoms selected from nitrogen, oxygen and sulfur. Whenever itappears herein, a numerical range such as “3 to 18” refers to eachinteger in the given range—e.g., “3 to 18 ring atoms” means that theheterocycloalkyl group may consist of 3 ring atoms, 4 ring atoms, etc.,up to and including 18 ring atoms. Unless stated otherwise specificallyin the specification, the heterocycloalkyl radical is a monocyclic,bicyclic, tricyclic or tetracyclic ring system, which may include fusedor bridged ring systems. The heteroatoms in the heterocycloalkyl radicalmay be optionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heterocycloalkyl radical is partially orfully saturated. The heterocycloalkyl may be attached to the rest of themolecule through any atom of the ring(s). Examples of suchheterocycloalkyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocycloalkyl moiety is optionally substituted byone or more substituents which independently are: alkyl, heteroalkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)N(R^(a))₂ (where t is 1 or2), or PO₃(R^(a))₂, where each R^(a) is independently hydrogen, alkyl,fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

“Heterocycloalkyl” also includes bicyclic ring systems wherein onenon-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2carbon atoms in addition to 1-3 heteroatoms independently selected fromoxygen, sulfur, and nitrogen, as well as combinations comprising atleast one of the foregoing heteroatoms; and the other ring, usually with3 to 7 ring atoms, optionally contains 1-3 heteroatoms independentlyselected from oxygen, sulfur, and nitrogen and is not aromatic.

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space—i.e., having a different stereochemical configuration.“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is a“racemic” mixture. The term “(±)” is used to designate a racemic mixturewhere appropriate. “Diastereoisomers” are stereoisomers that have atleast two asymmetric atoms, but which are not mirror-images of eachother. The absolute stereochemistry is specified according to theCahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer thestereochemistry at each chiral carbon can be specified by either R or S.Resolved compounds whose absolute configuration is unknown can bedesignated (+) or (−) depending on the direction (dextro- orlevorotatory) which they rotate plane polarized light at the wavelengthof the sodium D line. Certain of the compounds described herein containone or more asymmetric centers and can thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that can be defined, interms of absolute stereochemistry, as (R)- or (S)-. The present chemicalentities, pharmaceutical compositions and methods are meant to includeall such possible isomers, including racemic mixtures, optically pureforms and intermediate mixtures. Optically active (R)- and (S)-isomerscan be prepared using chiral synthons or chiral reagents, or resolvedusing conventional techniques. When the compounds described hereincontain olefinic double bonds or other centers of geometric asymmetry,and unless specified otherwise, it is intended that the compoundsinclude both E and Z geometric isomers.

“Enantiomeric purity” as used herein refers to the relative amounts,expressed as a percentage, of the presence of a specific enantiomerrelative to the other enantiomer. For example, if a compound, which maypotentially have an (R)- or an (S)-isomeric configuration, is present asa racemic mixture, the enantiomeric purity is about 50% with respect toeither the (R)- or (S)-isomer. If that compound has one isomeric formpredominant over the other, for example, 80% (S)- and 20% (R)-, theenantiomeric purity of the compound with respect to the (S)-isomericform is 80%. The enantiomeric purity of a compound can be determined ina number of ways known in the art, including but not limited tochromatography using a chiral support, polarimetric measurement of therotation of polarized light, nuclear magnetic resonance spectroscopyusing chiral shift reagents which include but are not limited tolanthanide containing chiral complexes or the Pirkle alcohol, orderivatization of a compounds using a chiral compound such as Mosher'sacid followed by chromatography or nuclear magnetic resonancespectroscopy.

“Moiety” refers to a specific segment or functional group of a molecule.Chemical moieties are often recognized chemical entities embedded in orappended to a molecule.

“Nitro” refers to the —NO₂ radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Tautomers” are structurally distinct isomers that interconvert bytautomerization. “Tautomerization” is a form of isomerization andincludes prototropic or proton-shift tautomerization, which isconsidered a subset of acid-base chemistry. “Prototropictautomerization” or “proton-shift tautomerization” involves themigration of a proton accompanied by changes in bond order, often theinterchange of a single bond with an adjacent double bond. Wheretautomerization is possible (e.g. in solution), a chemical equilibriumof tautomers can be reached. An example of tautomerization is keto-enoltautomerization. A specific example of keto-enol tautomerization is theinterconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-onetautomers. Another example of tautomerization is phenol-ketotautomerization. A specific example of phenol-keto tautomerization isthe interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.

The terms “enantiomerically enriched,” “enantiomerically pure,” and“non-racemic,” as used herein, refer to compositions in which thepercent by weight of one enantiomer is greater than the amount of thatone enantiomer in a control mixture of the racemic composition (e.g.,greater than 1:1 by weight). For example, an enantiomerically enrichedpreparation of the (S)-enantiomer, means a preparation of the compoundhaving greater than 50% by weight of the (S)-enantiomer relative to the(R)-enantiomer, such as at least 75% by weight, such as at least 80% byweight. In some embodiments, the enrichment can be significantly greaterthan 80% by weight, providing a “substantially enantiomericallyenriched,” “substantially enantiomerically pure,” or a “substantiallynon-racemic” preparation, which refers to preparations of compositionswhich have at least 85% by weight of one enantiomer relative to theother enantiomer, such as at least 90% by weight, and such as at least95% by weight. The terms “diastereomerically enriched” and“diastereomerically pure,” as used herein, refer to compositions inwhich the percent by weight of one diastereomer is greater than theamount of that one diastereomer in a control mixture of diastereomers.In some embodiments, the enrichment can be significantly greater than80% by weight, providing a “substantially diastereomerically enriched”or “substantially diastereomerically pure” preparation, which refers topreparations of compositions which have at least 85% by weight of onediastereomer relative to other diastereomers, such as at least 90% byweight, and such as at least 95% by weight.

In preferred embodiments, the enantiomerically enriched composition hasa higher potency with respect to therapeutic utility per unit mass thandoes the racemic mixture of that composition. Enantiomers can beisolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred enantiomerscan be prepared by asymmetric syntheses. See, for example, Jacques, etal., Enantiomers, Racemates and Resolutions, Wiley Interscience, NewYork (1981); E. L. Eliel and S. H. Wilen, Stereochemistry of OrganicCompounds, Wiley-Interscience, New York (1994).

A “leaving group or atom” is any group or atom that will, under selectedreaction conditions, cleave from the starting material, thus promotingreaction at a specified site. Examples of such groups, unless otherwisespecified, include halogen atoms and mesyloxy, p-nitrobenzensulphonyloxyand tosyloxy groups.

“Protecting group” is intended to mean a group that selectively blocksone or more reactive sites in a multifunctional compound such that achemical reaction can be carried out selectively on another unprotectedreactive site and the group can then be readily removed after theselective reaction is complete. A variety of protecting groups aredisclosed, for example, in T. H. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, New York(1999).

“Solvate” refers to a compound in physical association with one or moremolecules of a pharmaceutically acceptable solvent.

“Substituted” means that the referenced group may have attached one ormore additional moieties individually and independently selected from,for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl,carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy,aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester,thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo,perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl,sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- anddi-substituted amino groups, and protected derivatives thereof. Thesubstituents themselves may be substituted, for example, a cycloalkylsubstituent may itself have a halide substituent at one or more of itsring carbons.

“Sulfanyl” refers to groups that include —S-(optionally substitutedalkyl), —S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl) and —S-(optionally substituted heterocycloalkyl).

“Sulfinyl” refers to groups that include —S(O)—H, —S(O)-(optionallysubstituted alkyl), —S(O)-(optionally substituted amino),—S(O)-(optionally substituted aryl), —S(O)-(optionally substitutedheteroaryl) and —S(O)-(optionally substituted heterocycloalkyl).

“Sulfonyl” refers to groups that include —S(O₂)—H, —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-(optionally substituted amino),—S(O₂)-(optionally substituted aryl), —S(O₂)-(optionally substitutedheteroaryl), and —S(O₂)-(optionally substituted heterocycloalkyl).

“Sulfonamidyl” or “sulfonamido” refers to a —S(═O)₂—NRR radical, whereeach R is selected independently from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon). The R groups in —NRR ofthe —S(═O)₂—NRR radical may be taken together with the nitrogen to whichit is attached to form a 4-, 5-, 6- or 7-membered ring. A sulfonamidogroup is optionally substituted by one or more of the substituentsdescribed for alkyl, cycloalkyl, aryl, heteroaryl, respectively.

“Sulfoxyl” refers to a —S(═O)₂OH radical.

“Sulfonate” refers to a —S(═O)₂—OR radical, where R is selected from thegroup consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded througha ring carbon) and heteroalicyclic (bonded through a ring carbon). Asulfonate group is optionally substituted on R by one or more of thesubstituents described for alkyl, cycloalkyl, aryl, heteroaryl,respectively.

Compounds of the invention also include crystalline and amorphous formsof those compounds, including, for example, polymorphs,pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (includinganhydrates), conformational polymorphs, and amorphous forms of thecompounds, as well as mixtures thereof “Crystalline form” and“polymorph” are intended to include all crystalline and amorphous formsof the compound, including, for example, polymorphs, pseudopolymorphs,solvates, hydrates, unsolvated polymorphs (including anhydrates),conformational polymorphs, and amorphous forms, as well as mixturesthereof, unless a particular crystalline or amorphous form is referredto.

Compounds of the invention also include antibodies. The terms “antibody”and its plural form “antibodies” refer to whole immunoglobulins and anyantigen-binding fragment (“antigen-binding portion”) or single chainsthereof. An “antibody” further refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, C_(L). The V_(H) and V_(L) regions ofan antibody may be further subdivided into regions of hypervariability,which are referred to as complementarity determining regions (CDR) orhypervariable regions (HVR), and which can be interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen epitopeor epitopes. The constant regions of the antibodies may mediate thebinding of the immunoglobulin to host tissues or factors, includingvarious cells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The terms “monoclonal antibody,” “mAb,” “monoclonal antibodycomposition,” or their plural forms refer to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodycomposition displays a single binding specificity and affinity for aparticular epitope. Monoclonal antibodies specific to CD20 can be madeusing knowledge and skill in the art of injecting test subjects withCD20 antigen and then isolating hybridomas expressing antibodies havingthe desired sequence or functional characteristics. DNA encoding themonoclonal antibodies is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

The terms “antigen-binding portion” or “antigen-binding fragment” of anantibody (or simply “antibody portion”), as used herein, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen such as CD20. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) a Fd fragment consisting of the V_(H) and CH1 domains; (iv) a Fvfragment consisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a domain antibody (dAb) fragment (Ward et al., Nature,1989, 341, 544-546), which may consist of a V_(H) or a V_(L) domain; and(vi) an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules known as single chain Fv (scFv); see, for example, Bird etal., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad.Sci. USA 1988, 85, 5879-5883). Such scFv chain antibodies are alsointended to be encompassed within the terms “antigen-binding portion” or“antigen-binding fragment” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

The term “human antibody,” as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). The term “human antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody. The term “conjugate” or “immunoconjugate” refers to anantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a bacterial toxin, a cytotoxic drug or a radionuclide-containingtoxin. Toxic moieties can be conjugated to antibodies of the inventionusing methods available in the art.

The terms “humanized antibody,” “humanized antibodies,” and “humanized”are intended to refer to antibodies in which CDR sequences derived fromthe germline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences. Additional framework regionmodifications may be made within the human framework sequences.Humanized forms of non-human (for example, murine) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from a 15hypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature 1986, 321,522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr.Op. Struct. Biol. 1992, 2, 593-596.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

A “diabody” is a small antibody fragment with two antigen-binding sites.The fragment comprises a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L) or V_(L)-V_(H)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,e.g., European Patent No. EP 404,097, International Patent PublicationNo. WO 93/11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993,90, 6444-6448.

The term “glycosylation” refers to a modified derivative of an antibody.An aglycoslated antibody lacks glycosylation. Glycosylation can bealtered to, for example, increase the affinity of the antibody forantigen. Such carbohydrate modifications can be accomplished by, forexample, altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Aglycosylation may increase the affinity of the antibody for antigen, asdescribed in U.S. Pat. Nos. 5,714,350 and 6,350,861. Additionally oralternatively, an antibody can be made that has an altered type ofglycosylation, such as a hypofucosylated antibody having reduced amountsof fucosyl residues or an antibody having increased bisecting GlcNacstructures. Such altered glycosylation patterns have been demonstratedto increase the ability of antibodies. Such carbohydrate modificationscan be accomplished by, for example, expressing the antibody in a hostcell with altered glycosylation machinery. Cells with alteredglycosylation machinery have been described in the art and can be usedas host cells in which to express recombinant antibodies of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), suchthat antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8−/− celllines were created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see e.g. U.S. PatentPublication No. 2004/0110704 or Yamane-Ohnuki, et al., Biotechnol.Bioeng., 2004, 87, 614-622). As another example, European Patent No. EP1,176,195 describes a cell line with a functionally disrupted FUT8 gene,which encodes a fucosyl transferase, such that antibodies expressed insuch a cell line exhibit hypofucosylation by reducing or eliminating thealpha 1,6 bond-related enzyme, and also describes cell lines which havea low enzyme activity for adding fucose to the N-acetylglucosamine thatbinds to the Fc region of the antibody or does not have the enzymeactivity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).International Patent Publication WO 03/035835 describes a variant CHOcell line, Lec 13 cells, with reduced ability to attach fucose toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (see also Shields, et al., J.Biol. Chem. 2002, 277, 26733-26740. International Patent Publication WO99/54342 describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana, et al., Nat. Biotech. 1999, 17,176-180). Alternatively, the fucose residues of the antibody may becleaved off using a fucosidase enzyme. For example, the fucosidasealpha-L-fucosidase removes fucosyl residues from antibodies as describedin Tarentino, et al., Biochem. 1975, 14, 5516-5523.

“Pegylation” refers to a modified antibody, or a fragment thereof, thattypically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Pegylation may, for example, increase the biological (e.g., serum) halflife of the antibody. Preferably, the pegylation is carried out via anacylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C₁-C₁₀) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. The antibody to be pegylated may be an aglycosylatedantibody. Methods for pegylation are known in the art and can be appliedto the antibodies of the invention. See, for example, European PatentNos. EP 0154316 and EP 0401384.

As used herein, an antibody that “specifically binds to human CD20” isintended to refer to an antibody that binds to human CD20 with a K_(D)of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, more preferably1×10⁻⁸ M or less, more preferably 5×10⁻⁹ M or less.

The term “radioisotope-labeled complex” refers to both non-covalent andcovalent attachment of a radioactive isotope, such as ⁹⁰Y, ¹¹¹In, or¹³¹I, to an antibody.

The term “biosimilar” means a biological product that is highly similarto a U.S. licensed reference biological product notwithstanding minordifferences in clinically inactive components, and for which there areno clinically meaningful differences between the biological product andthe reference product in terms of the safety, purity, and potency of theproduct. Furthermore, a similar biological or “biosimilar” medicine is abiological medicine that is similar to another biological medicine thathas already been authorized for use by the European Medicines Agency.The term “biosimilar” is also used synonymously by other national andregional regulatory agencies. Biological products or biologicalmedicines are medicines that are made by or derived from a biologicalsource, such as a bacterium or yeast. They can consist of relativelysmall molecules such as human insulin or erythropoietin, or complexmolecules such as monoclonal antibodies. For example, if the referenceanti-CD20 monoclonal antibody is rituximab, an anti-CD20 biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to rituximab is a “biosimilar to” rituximab or is a“biosimilar thereof” rituximab.

BTK Inhibitors

In an embodiment, the BTK inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,wherein:

X is CH, N, O or S; Y is C(R₆), N, O or S;

Z is CH, N or bond;

A is CH or N;

B₁ is N or C(R₇);B₂ is N or C(R₈);B₃ is N or C(R₉);B₄ is N or C(R₁₀);R₁ is R₁₁C(═O), R₁₂S(═O), R₁₃S(═O)₂ or (C₁₋₆)alkyl optionallysubstituted with R₁₄;R₂ is H, (C₁₋₃)alkyl or (C₃₋₇)cycloalkyl;R₃ is H, (C₁₋₆)alkyl or (C₃₋₇)cycloalkyl); orR₂ and R₃ form, together with the N and C atom they are attached to, a(C₃₋₇)heterocycloalkyl optionally substituted with one or more fluorine,hydroxyl, (C₁₋₃)alkyl, (C₁₋₃)alkoxy or oxo;R₄ is H or (C₁₋₃)alkyl;R₅ is H, halogen, cyano, (C₁₋₄)alkyl, (C₁₋₃)alkoxy, (C₃₋₆)cycloalkyl,any alkyl group of which is optionally substituted with one or morehalogen; or R₅ is (C₆₋₁₀)aryl or (C₂₋₆) heterocycloalkyl;R₆ is H or (C₁₋₃)alkyl; orR₅ and R₆ together may form a (C₃₋₇)cycloalkenyl or(C₂₋₆)heterocycloalkenyl, each optionally substituted with (C₁₋₃)alkylor one or more halogens;R₇ is H, halogen, CF₃, (C₁₋₃)alkyl or (C₁₋₃)alkoxy;R₈ is H, halogen, CF₃, (C₁₋₃)alkyl or (C₁₋₃)alkoxy; orR₇ and R₈ together with the carbon atoms they are attached to, form(C₆₋₁₀)aryl or (C₁₋₉) heteroaryl;R₉ is H, halogen, (C₁₋₃)alkyl or (C₁₋₃)alkoxy;R₁₀ is H, halogen, (C₁₋₃)alkyl or (C₁₋₃)alkoxy;R₁₁ is independently selected from the group consisting of (C₁₋₆)alkyl,(C₂₋₆)alkenyl and (C₂₋₆)alkynyl, where each alkyl, alkenyl or alkynyl isoptionally substituted with one or moresubstituents selected from the group consisting of hydroxyl,(C₁₋₄)alkyl, (C₃₋₇)cycloalkyl, [(C₁₋₄)alkyl]amino, di[(C₁₋₄)alkyl]amino,(C₁₋₃)alkoxy, (C₃₋₇)cycloalkoxy, (C₆₋₁₀)aryl and (C₃₋₇)heterocycloalkyl;or R₁₁ is (C₁₋₃)alkyl-C(O)—S—(C₁₋₃)alkyl; orR₁₁ is (C₁₋₅)heteroaryl optionally substituted with one or moresubstituents selected from the group consisting of halogen or cyano;R₁₂ and R₁₃ are independently selected from the group consisting of(C₂₋₆)alkenyl or (C₂₋₆) alkynyl, both optionally substituted with one ormore substituents selected from the group consisting of hydroxyl,(C₁₋₄)alkyl, (C₃₋₇)cycloalkyl, [(C₁₋₄)alkyl]amino, di[(C₁₋₄)alkyl]amino,(C₁₋₃)alkoxy, (C₃₋₇)cycloalkoxy, (C₆₋₁₀)aryl and (C₃₋₇)heterocycloalkyl;or a (C₁₋₅)heteroaryl optionally substituted with one or moresubstituents selected from the group consisting of halogen and cyano;andR₁₄ is independently selected from the group consisting of halogen,cyano, (C₂₋₆)alkenyl and (C₂₋₆)alkynyl, both optionally substituted withone or more substituents selected from the group consisting of hydroxyl,(C₁₋₄)alkyl, (C₃₋₇)cycloalkyl, (C₁₋₄)alkylamino, di[(C₁₋₄)alkyl]amino,(C₁₋₃)alkoxy, (C₃₋₇)cycloalkoxy, (C₆₋₁₀)aryl, (C₁₋₅)heteroaryl and(C₃₋₇)heterocycloalkyl;with the proviso that:0 to 2 atoms of X, Y, Z can simultaneously be a heteroatom;when one atom selected from X, Y is O or S, then Z is a bond and theother atom selected from X, Y can not be O or S;when Z is C or N then Y is C(R₆) or N and X is C or N;0 to 2 atoms of B₁, B₂, B₃, and B₄ are N;with the terms used having the following meanings:(C₁₋₂)alkyl means an alkyl group having 1 to 2 carbon atoms, beingmethyl or ethyl,(C₁₋₃)alkyl means a branched or unbranched alkyl group having 1-3 carbonatoms, being methyl, ethyl, propyl or isopropyl;(C₁₋₄)alkyl means a branched or unbranched alkyl group having 1-4 carbonatoms, being methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl and tert-butyl, (C₁₋₃)alkyl groups being preferred;(C₁₋₅)alkyl means a branched or unbranched alkyl group having 1-5 carbonatoms, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl and isopentyl, (C₁₋₄)alkyl groups beingpreferred. (C₁₋₆)Alkyl means a branched or unbranched alkyl group having1-6 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl,tert-butyl, n-pentyl and n-hexyl. (C₁₋₅)alkyl groups are preferred,(C₁₋₄)alkyl being most preferred;(C₁₋₂)alkoxy means an alkoxy group having 1-2 carbon atoms, the alkylmoiety having the same meaning as previously defined;(C₁₋₃)alkoxy means an alkoxy group having 1-3 carbon atoms, the alkylmoiety having the same meaning as previously defined. (C₁₋₂)alkoxygroups are preferred;(C₁₋₄)alkoxy means an alkoxy group having 1-4 carbon atoms, the alkylmoiety having the same meaning as previously defined. (C₁₋₃)alkoxygroups are preferred, (C₁₋₂)alkoxy groups being most preferred;(C₂₋₄)alkenyl means a branched or unbranched alkenyl group having 2-4carbon atoms, such as ethenyl, 2-propenyl, isobutenyl or 2-butenyl;(C₂₋₆)alkenyl means a branched or unbranched alkenyl group having 2-6carbon atoms, such as ethenyl, 2-butenyl, and n-pentenyl, (C₂₋₄)alkenylgroups being most preferred;(C₂₋₄)alkynyl means a branched or unbranched alkynyl group having 2-4carbon atoms, such as ethynyl, 2-propynyl or 2-butynyl;(C₂₋₆)alkynyl means a branched or unbranched alkynyl group having 2-6carbon atoms, such as ethynyl, propynyl, n-butynyl, n-pentynyl,isopentynyl, isohexynyl or n-hexynyl. (C₂₋₄)alkynyl groups arepreferred; (C₃₋₆)cycloalkyl means a cycloalkyl group having 3-6 carbonatoms, being cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;(C₃₋₇)cycloalkyl means a cycloalkyl group having 3-7 carbon atoms, beingcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl;(C₂₋₆)heterocycloalkyl means a heterocycloalkyl group having 2-6 carbonatoms, preferably 3-5 carbon atoms, and one or two heteroatoms selectedfrom N, O and/or S, which may be attached via a heteroatom if feasible,or a carbon atom; preferred heteroatoms are N or O; also preferred arepiperidine, morpholine, pyrrolidine and piperazine; with the mostpreferred (C₂₋₆)heterocycloalkyl being pyrrolidine; the heterocycloalkylgroup may be attached via a heteroatom if feasible;(C₃₋₇)heterocycloalkyl means a heterocycloalkyl group having 3-7 carbonatoms, preferably 3-5 carbon atoms, and one or two heteroatoms selectedfrom N, O and/or S. Preferred heteroatoms are N or O; preferred (C₃₋₇)heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl,homopiperidinyl or morpholinyl; more preferred (C₃₋₇)heterocycloalkylgroups are piperidine, morpholine and pyrrolidine; and theheterocycloalkyl group may be attached via a heteroatom if feasible;(C₃₋₇)cycloalkoxy means a cycloalkyl group having 3-7 carbon atoms, withthe same meaning as previously defined, attached via a ring carbon atomto an exocyclic oxygen atom;(C₆₋₁₀)aryl means an aromatic hydrocarbon group having 6-10 carbonatoms, such as phenyl, naphthyl, tetrahydronaphthyl or indenyl; thepreferred (C₆₋₁₀)aryl group is phenyl;(C₁₋₅)heteroaryl means a substituted or unsubstituted aromatic grouphaving 1-5 carbon atoms and 1-4 heteroatoms selected from N, O and/or S;the (C₁₋₅)heteroaryl may optionally be substituted; preferred(C₁₋₅)heteroaryl groups are tetrazolyl, imidazolyl, thiadiazolyl,pyridyl, pyrimidyl, triazinyl, thienyl or furyl, a more preferred(C₁₋₅)heteroaryl is pyrimidyl;(C₁₋₉)heteroaryl means a substituted or unsubstituted aromatic grouphaving 1-9 carbon atoms and 1-4 heteroatoms selected from N, O and/or S;the (C₁₋₉)heteroaryl may optionally be substituted; preferred(C₁₋₉)heteroaryl groups are quinoline, isoquinoline and indole;[(C₁₋₄)alkyl]amino means an amino group, monosubstituted with an alkylgroup containing 1-4 carbon atoms having the same meaning as previouslydefined; preferred [(C₁₋₄)alkyl]amino group is methylamino;di[(C₁₋₄)alkyl]amino means an amino group, disubstituted with alkylgroup(s), each containing 1-4 carbon atoms and having the same meaningas previously defined; preferred di[(C₁₋₄)alkyl]amino group isdimethylamino;halogen means fluorine, chlorine, bromine or iodine;(C₁₋₃)alkyl-C(O)—S—(C₁₋₃)alkyl means an alkyl-carbonyl-thio-alkyl group,each of the alkyl groups having 1 to 3 carbon atoms with the samemeaning as previously defined;(C₃₋₇)cycloalkenyl means a cycloalkenyl group having 3-7 carbon atoms,preferably 5-7 carbon atoms; preferred (C₃₋₇)cycloalkenyl groups arecyclopentenyl or cyclohexenyl; cyclohexenyl groups are most preferred;(C₂₋₆)heterocycloalkenyl means a heterocycloalkenyl group having 2-6carbon atoms, preferably 3-5 carbon atoms; and 1 heteroatom selectedfrom N, O and/or S; preferred (C₂₋₆)heterocycloalkenyl groups areoxycyclohexenyl and azacyclohexenyl group.In the above definitions with multifunctional groups, the attachmentpoint is at the last group.When, in the definition of a substituent, is indicated that “all of thealkyl groups” of said substituent are optionally substituted, this alsoincludes the alkyl moiety of an alkoxy group.A circle in a ring of Formula (I) indicates that the ring is aromatic.Depending on the ring formed, the nitrogen, if present in X or Y, maycarry a hydrogen.The term “substituted” means that one or more hydrogens on thedesignated atom/atoms is/are replaced with a selection from theindicated group, provided that the designated atom's normal valencyunder the existing circumstances is not exceeded, and that thesubstitution results in a stable compound. Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds. “Stable compound” or “stable structure” is defined asa compound or structure that is sufficiently robust to survive isolationto a useful degree of purity from a reaction mixture, and formulationinto a drug product containing an efficacious active pharmaceuticalingredient.The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

In an embodiment of Formula (I), B₁ is C(R₇); B₂ is C(R₈); B₃ is C(R₉);B₄ is C(R₁₀); R₇, R₉, and R₁₀ are each H; and R₈ is hydrogen or methyl.

In an embodiment of Formula (I), the ring containing X, Y and Z isselected from the group consisting of pyridyl, pyrimidyl, pyridazyl,triazinyl, thiazolyl, oxazolyl and isoxazolyl.

In an embodiment of Formula (I), the ring containing X, Y and Z isselected from the group consisting of pyridyl, pyrimidyl and pyridazyl.

In an embodiment of Formula (I), the ring containing X, Y and Z isselected from the group consisting of pyridyl and pyrimidyl.

In an embodiment of Formula (I), the ring containing X, Y and Z ispyridyl.

In an embodiment of Formula (I), R₅ is selected from the groupconsisting of hydrogen, fluorine, methyl, methoxy and trifluoromethyl.

In an embodiment of Formula (I), R₅ is hydrogen.

In an embodiment of Formula (I), R₂ and R₃ together form aheterocycloalkyl ring selected from the group consisting of azetidinyl,pyrrolidinyl, piperidinyl, homopiperidinyl and morpholinyl, optionallysubstituted with one or more of fluoro, hydroxyl, (C₁₋₃)alkyl and(C₁₋₃)alkoxy.

In an embodiment of Formula (I), R₂ and R₃ together form aheterocycloalkyl ring selected from the group consisting of azetidinyl,pyrrolidinyl and piperidinyl.

In an embodiment of Formula (I), R₂ and R₃ together form a pyrrolidinylring.

In an embodiment of Formula (I), R₁ is independently selected from thegroup consisting of (C₁₋₆)alkyl, (C₂₋₆)alkenyl or (C₂₋₆)alkynyl, eachoptionally substituted with one or more substituents selected from thegroup consisting of hydroxyl, (C₁₋₄)alkyl, (C₃₋₇)cycloalkyl,[(C₁₋₄)alkyl]amino, di[(C₁₋₄)alkyl]amino, (C₁₋₃)alkoxy,(C₃₋₇)cycloalkoxy, (C₆₋₁₀)aryl and (C₃₋₇)heterocycloalkyl.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X is N; Y andZ are CH; R₅ is CH₃; A is N; R₂, R₃ and R₄ are H; and R₁ is CO—CH₃.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X and Y areN; Z is CH; R₅ is CH₃; A is N; R₂, R₃ and R₄ are H; and R₁ is CO—CH₃.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X and Y areN; Z is CH; R₅ is CH₃; A is CH; R₂ and R₃ together form a piperidinylring; R₄ is H; and R₁ is CO-ethenyl.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X, Y and Zare CH; R₅ is H; A is CH; R₂ and R₃ together form a pyrrolidinyl ring;R₄ is H; and R₁ is CO-propynyl.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X, Y and Zare CH; R₅ is CH₃; A is CH; R₂ and R₃ together form a piperidinyl ring;R₄ is H; and R₁ is CO-propynyl.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X and Y areN; Z is CH; R₅ is H; A is CH; R₂ and R₃ together form a morpholinylring; R₄ is H; and R₁ is CO-ethenyl.

In an embodiment of Formula (I), B₁, B₂, B₃ and B₄ are CH; X and Y areN; Z is CH; R₅ is CH₃; A is CH; R₂ and R₃ together form a morpholinylring; R₄ is H; and R₁ is CO-propynyl.

In a preferred embodiment, the BTK inhibitor is a compound of Formula(II):

or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The compound of Formula (II) is also known as(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamide.In an embodiment, the BTK inhibitor is(S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamideor a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, orprodrug thereof. The preparation of this compound is described atExample 6 of U.S. Patent Application Publication No. US 2014/0155385 A1,the disclosure of which is incorporated herein by reference. Briefly,the preparation of Formula (II) can be accomplished by the followingprocedure.1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (also known as HATU,N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide, andO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate)(18.75 mg, 0.049 mmol) was added to a solution of(S)-4-(8-amino-3-(pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamide(19.7 mg, 0.049 mmol), triethylamine (20 mg, 0.197 mmol, 0.027 mL) and2-butynoic acid in dichloromethane (2 mL). The mixture was stirred for30 minutes at room temperature. The mixture was washed with water driedover magnesium sulfate and concentrated under vacuum. The residue waspurified by preparative liquid chromatography. Fractions containingproduct were collected and reduced to dryness to afford 10.5 mg ofFormula (II) (18.0% yield).

Pharmaceutical Compositions

In selected embodiments, the invention provides pharmaceuticalcompositions for treating lymphoma and leukemia, including CLL and SLL.

The pharmaceutical compositions are typically formulated to provide atherapeutically effective amount of a BTK inhibitor, including the BTKinhibitors of Formula (I) or Formula (II), or a pharmaceuticallyacceptable salt, ester, prodrug, solvate, hydrate or derivative thereof.Where desired, the pharmaceutical compositions contain apharmaceutically acceptable salt and/or coordination complex thereof,and one or more pharmaceutically acceptable excipients, carriers,including inert solid diluents and fillers, diluents, including sterileaqueous solution and various organic solvents, permeation enhancers,solubilizers and adjuvants. Where desired, other active ingredients inaddition to a BTK inhibitor of Formula (I) or Formula (II) may be mixedinto a preparation or both components may be formulated into separatepreparations for use in combination separately or at the same time.

In selected embodiments, the concentration of the BTK inhibitors ofFormula (I) or Formula (II) provided in the pharmaceutical compositionsof the invention is less than, for example, 100%, 90%, 80%, 70%, 60%,50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v or v/v.

In selected embodiments, the concentration of the BTK inhibitors ofFormula (I) or Formula (II) provided in the pharmaceutical compositionsof the invention is independently greater than 90%, 80%, 70%, 60%, 50%,40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%,17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%,15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%,12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%,10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%,7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%,4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%,1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%,0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%,0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%,0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w,w/v, or v/v.

In selected embodiments, the concentration of the BTK inhibitors ofFormula (I) or Formula (II) is independently in the range fromapproximately 0.0001% to approximately 50%, approximately 0.001% toapproximately 40%, approximately 0.01% to approximately 30%,approximately 0.02% to approximately 29%, approximately 0.03% toapproximately 28%, approximately 0.04% to approximately 27%,approximately 0.05% to approximately 26%, approximately 0.06% toapproximately 25%, approximately 0.07% to approximately 24%,approximately 0.08% to approximately 23%, approximately 0.09% toapproximately 22%, approximately 0.1% to approximately 21%,approximately 0.2% to approximately 20%, approximately 0.3% toapproximately 19%, approximately 0.4% to approximately 18%,approximately 0.5% to approximately 17%, approximately 0.6% toapproximately 16%, approximately 0.7% to approximately 15%,approximately 0.8% to approximately 14%, approximately 0.9% toapproximately 12% or approximately 1% to approximately 10% w/w, w/v orv/v.

In selected embodiments, the concentration of the BTK inhibitors ofFormula (I) or Formula (II) is independently in the range fromapproximately 0.001% to approximately 10%, approximately 0.01% toapproximately 5%, approximately 0.02% to approximately 4.5%,approximately 0.03% to approximately 4%, approximately 0.04% toapproximately 3.5%, approximately 0.05% to approximately 3%,approximately 0.06% to approximately 2.5%, approximately 0.07% toapproximately 2%, approximately 0.08% to approximately 1.5%,approximately 0.09% to approximately 1%, approximately 0.1% toapproximately 0.9% w/w, w/v or v/v.

In selected embodiments, the amount of the BTK inhibitors of Formula (I)or Formula (II) is independently equal to or less than 10 g, 9.5 g, 9.0g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g,3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g,0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g,0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g,0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In selected embodiments, the amount of the BTK inhibitors of Formula (I)or Formula (II) is independently more than 0.0001 g, 0.0002 g, 0.0003 g,0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g,0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g,0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g,0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g,0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g,5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

The BTK inhibitors of Formula (I) or Formula (II) are effective over awide dosage range. For example, in the treatment of adult humans,dosages independently ranging from 0.01 to 1000 mg, from 0.5 to 100 mg,from 1 to 50 mg per day, and from 5 to 40 mg per day are examples ofdosages that may be used. The exact dosage will depend upon the route ofadministration, the form in which the compound is administered, thegender and age of the subject to be treated, the body weight of thesubject to be treated, and the preference and experience of theattending physician.

Described below are non-limiting exemplary pharmaceutical compositionsand methods for preparing the same.

Pharmaceutical Compositions for Oral Administration

In selected embodiments, the invention provides a pharmaceuticalcomposition for oral administration containing a BTK inhibitor ofFormula (I) or Formula (II), and a pharmaceutical excipient suitable fororal administration.

In selected embodiments, the invention provides a solid pharmaceuticalcomposition for oral administration containing: (i) an effective amountof a BTK inhibitor of Formula (I) or Formula (II), in combination and(ii) a pharmaceutical excipient suitable for oral administration. Inselected embodiments, the composition further contains (iii) aneffective amount of at least one additional active ingredient.

In selected embodiments, the pharmaceutical composition may be a liquidpharmaceutical composition suitable for oral consumption. Pharmaceuticalcompositions of the invention suitable for oral administration can bepresented as discrete dosage forms, such as capsules, cachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. Such dosage forms can beprepared by any of the methods of pharmacy, but all methods include thestep of bringing the active ingredient(s) into association with thecarrier, which constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient(s) with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product intothe desired presentation. For example, a tablet can be prepared bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets can be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such aspowder or granules, optionally mixed with an excipient such as, but notlimited to, a binder, a lubricant, an inert diluent, and/or a surfaceactive or dispersing agent. Molded tablets can be made by molding in asuitable machine a mixture of the powdered compound moistened with aninert liquid diluent.

The invention further encompasses anhydrous pharmaceutical compositionsand dosage forms since water can facilitate the degradation of somecompounds. For example, water may be added (e.g., 5%) in thepharmaceutical arts as a means of simulating long-term storage in orderto determine characteristics such as shelf-life or the stability offormulations over time. Anhydrous pharmaceutical compositions and dosageforms of the invention can be prepared using anhydrous or low moisturecontaining ingredients and low moisture or low humidity conditions.Pharmaceutical compositions and dosage forms of the invention whichcontain lactose can be made anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected. An anhydrous pharmaceutical composition may be prepared andstored such that its anhydrous nature is maintained. Accordingly,anhydrous compositions may be packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastic or the like, unit dose containers,blister packs, and strip packs.

The BTK inhibitors of Formula (I) or Formula (II) can be combined in anintimate admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier can takea wide variety of forms depending on the form of preparation desired foradministration. In preparing the compositions for an oral dosage form,any of the usual pharmaceutical media can be employed as carriers, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (such as suspensions, solutions, and elixirs) or aerosols;or carriers such as starches, sugars, micro-crystalline cellulose,diluents, granulating agents, lubricants, binders, and disintegratingagents can be used in the case of oral solid preparations, in someembodiments without employing the use of lactose. For example, suitablecarriers include powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Binders suitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixturesthereof.

Examples of suitable fillers for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Too much of a disintegrant may produce tablets whichdisintegrate in the bottle. Too little may be insufficient fordisintegration to occur, thus altering the rate and extent of release ofthe active ingredients from the dosage form. Thus, a sufficient amountof disintegrant that is neither too little nor too much to detrimentallyalter the release of the active ingredient(s) may be used to form thedosage forms of the compounds disclosed herein. The amount ofdisintegrant used may vary based upon the type of formulation and modeof administration, and may be readily discernible to those of ordinaryskill in the art. About 0.5 to about 15 weight percent of disintegrant,or about 1 to about 5 weight percent of disintegrant, may be used in thepharmaceutical composition. Disintegrants that can be used to formpharmaceutical compositions and dosage forms of the invention include,but are not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oraladministration, the essential active ingredient therein may be combinedwith various sweetening or flavoring agents, coloring matter or dyesand, if so desired, emulsifying and/or suspending agents, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

Surfactants which can be used to form pharmaceutical compositions anddosage forms of the invention include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants may be employed, a mixtureof lipophilic surfactants may be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant may beemployed.

A suitable hydrophilic surfactant may generally have an HLB value of atleast 10, while suitable lipophilic surfactants may generally have anHLB value of or less than about 10. An empirical parameter used tocharacterize the relative hydrophilicity and hydrophobicity of non-ionicamphiphilic compounds is the hydrophilic-lipophilic balance (“HLB”value). Surfactants with lower HLB values are more lipophilic orhydrophobic, and have greater solubility in oils, while surfactants withhigher HLB values are more hydrophilic, and have greater solubility inaqueous solutions. Hydrophilic surfactants are generally considered tobe those compounds having an HLB value greater than about 10, as well asanionic, cationic, or zwitterionic compounds for which the HLB scale isnot generally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts;fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides; glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof;carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate; acylactylates; mono- and di-acetylated tartaricacid esters of mono- and di-glycerides; succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, ionic surfactants include, by way ofexample: lecithins, lysolecithin, phospholipids, lysophospholipids andderivatives thereof; carnitine fatty acid ester salts; salts ofalkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; and mixtures thereof.

Ionic surfactants may be the ionized forms of lecithin, lysolecithin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidic acid, phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides, cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, andsalts and mixtures thereof.

Hydrophilic non-ionic surfactants may include, but not limited to,alkylglucosides; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylenesterols, derivatives, and analogues thereof; polyoxyethylated vitaminsand derivatives thereof; polyoxyethylene-polyoxypropylene blockcopolymers; and mixtures thereof; polyethylene glycol sorbitan fattyacid esters and hydrophilic transesterification products of a polyolwith at least one member of the group consisting of triglycerides,vegetable oils, and hydrogenated vegetable oils. The polyol may beglycerol, ethylene glycol, polyethylene glycol, sorbitol, propyleneglycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate,PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol,polyglyceryl-10oleate, Tween 40, Tween 60, sucrose monostearate, sucrosemonolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG15-100 octyl phenol series, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fattyalcohols; glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers; sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides;hydrophobic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and mixtures thereof. Within this group,preferred lipophilic surfactants include glycerol fatty acid esters,propylene glycol fatty acid esters, and mixtures thereof, or arehydrophobic transesterification products of a polyol with at least onemember of the group consisting of vegetable oils, hydrogenated vegetableoils, and triglycerides.

In an embodiment, the composition may include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentinvention and to minimize precipitation of the compound of the presentinvention. This can be especially important for compositions fornon-oral use, such as for compositions for injection. A solubilizer mayalso be added to increase the solubility of the hydrophilic drug and/orother components, such as surfactants, or to maintain the composition asa stable or homogeneous solution or dispersion.

Examples of suitable solubilizers include, but are not limited to, thefollowing: alcohols and polyols, such as ethanol, isopropanol, butanol,benzyl alcohol, ethylene glycol, propylene glycol, butanediols andisomers thereof, glycerol, pentaerythritol, sorbitol, mannitol,transcutol, dimethyl isosorbide, polyethylene glycol, polypropyleneglycol, polyvinylalcohol, hydroxypropyl methylcellulose and othercellulose derivatives, cyclodextrins and cyclodextrin derivatives;ethers of polyethylene glycols having an average molecular weight ofabout 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether(glycofurol) or methoxy PEG; amides and other nitrogen-containingcompounds such as 2-pyrrolidone, 2-piperidone, E-caprolactam,N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone,N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esterssuch as ethyl propionate, tributylcitrate, acetyl triethylcitrate,acetyl tributyl citrate, triethylcitrate, ethyl oleate, ethyl caprylate,ethyl butyrate, triacetin, propylene glycol monoacetate, propyleneglycol diacetate, .epsilon.-caprolactone and isomers thereof,6-valerolactone and isomers thereof, β-butyrolactone and isomersthereof; and other solubilizers known in the art, such as dimethylacetamide, dimethyl isosorbide, N-methyl pyrrolidones, monooctanoin,diethylene glycol monoethyl ether, and water.

Mixtures of solubilizers may also be used. Examples include, but notlimited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate,dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol,transcutol, propylene glycol, and dimethyl isosorbide. Particularlypreferred solubilizers include sorbitol, glycerol, triacetin, ethylalcohol, PEG-400, glycofurol and propylene glycol.

The amount of solubilizer that can be included is not particularlylimited. The amount of a given solubilizer may be limited to abioacceptable amount, which may be readily determined by one of skill inthe art. In some circumstances, it may be advantageous to includeamounts of solubilizers far in excess of bioacceptable amounts, forexample to maximize the concentration of the drug, with excesssolubilizer removed prior to providing the composition to a patientusing conventional techniques, such as distillation or evaporation.Thus, if present, the solubilizer can be in a weight ratio of 10%, 25%,50%, 100%, or up to about 200% by weight, based on the combined weightof the drug, and other excipients. If desired, very small amounts ofsolubilizer may also be used, such as 5%, 2%, 1% or even less.Typically, the solubilizer may be present in an amount of about 1% toabout 100%, more typically about 5% to about 25% by weight.

The composition can further include one or more pharmaceuticallyacceptable additives and excipients. Such additives and excipientsinclude, without limitation, detackifiers, anti-foaming agents,buffering agents, polymers, antioxidants, preservatives, chelatingagents, viscomodulators, tonicifiers, flavorants, colorants, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof.

In addition, an acid or a base may be incorporated into the compositionto facilitate processing, to enhance stability, or for other reasons.Examples of pharmaceutically acceptable bases include amino acids, aminoacid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide,sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate,magnesium hydroxide, magnesium aluminum silicate, synthetic aluminumsilicate, synthetic hydrocalcite, magnesium aluminum hydroxide,diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine,triethylamine, triisopropanolamine, trimethylamine,tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable arebases that are salts of a pharmaceutically acceptable acid, such asacetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonicacid, amino acids, ascorbic acid, benzoic acid, boric acid, butyricacid, carbonic acid, citric acid, fatty acids, formic acid, fumaricacid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lacticacid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionicacid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinicacid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonicacid, uric acid, and the like. Salts of polyprotic acids, such as sodiumphosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphatecan also be used. When the base is a salt, the cation can be anyconvenient and pharmaceutically acceptable cation, such as ammonium,alkali metals and alkaline earth metals. Examples may include, but arenot limited to, sodium, potassium, lithium, magnesium, calcium andammonium.

Suitable acids are pharmaceutically acceptable organic or inorganicacids. Examples of suitable inorganic acids include hydrochloric acid,hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boricacid, phosphoric acid, and the like. Examples of suitable organic acidsinclude acetic acid, acrylic acid, adipic acid, alginic acid,alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boricacid, butyric acid, carbonic acid, citric acid, fatty acids, formicacid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbicacid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid,para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid,salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid,thioglycolic acid, toluenesulfonic acid and uric acid.

Pharmaceutical Compositions for Injection

In selected embodiments, the invention provides a pharmaceuticalcomposition for injection containing a BTK inhibitor of Formula (I) orFormula (II), and a pharmaceutical excipient suitable for injection.Components and amounts of agents in the compositions are as describedherein.

The forms in which the compositions of the present invention may beincorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (andsuitable mixtures thereof), cyclodextrin derivatives, and vegetable oilsmay also be employed. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, for the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid and thimerosal.

Sterile injectable solutions are prepared by incorporating a BTKinhibitor of Formula (I) or Formula (II) in the required amounts in theappropriate solvent with various other ingredients as enumerated above,as required, followed by filtered sterilization. Generally, dispersionsare prepared by incorporating the various sterilized active ingredientsinto a sterile vehicle which contains the basic dispersion medium andthe required other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,certain desirable methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Pharmaceutical Compositions for Topical Delivery

In some embodiments, the invention provides a pharmaceutical compositionfor transdermal delivery containing the BTK inhibitors of Formula (I) orFormula (II) and a pharmaceutical excipient suitable for transdermaldelivery.

Compositions of the present invention can be formulated intopreparations in solid, semi-solid, or liquid forms suitable for local ortopical administration, such as gels, water soluble jellies, creams,lotions, suspensions, foams, powders, slurries, ointments, solutions,oils, pastes, suppositories, sprays, emulsions, saline solutions,dimethylsulfoxide (DMSO)-based solutions. In general, carriers withhigher densities are capable of providing an area with a prolongedexposure to the active ingredients. In contrast, a solution formulationmay provide more immediate exposure of the active ingredient to thechosen area.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients, which are compounds that allow increasedpenetration of, or assist in the delivery of, therapeutic moleculesacross the stratum corneum permeability barrier of the skin. There aremany of these penetration-enhancing molecules known to those trained inthe art of topical formulation. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, water, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Another exemplary formulation for use in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the BTK inhibitors of Formula (I) or Formula (II) incontrolled amounts, either with or without another active pharmaceuticalingredient.

The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, e.g., U.S. Pat.Nos. 5,023,252; 4,992,445 and 5,001,139. Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

Other Pharmaceutical Compositions

Pharmaceutical compositions may also be prepared from compositionsdescribed herein and one or more pharmaceutically acceptable excipientssuitable for sublingual, buccal, rectal, intraosseous, intraocular,intranasal, epidural, or intraspinal administration. Preparations forsuch pharmaceutical compositions are well-known in the art. See, e.g.,Anderson, et al. eds., Handbook of Clinical Drug Data, Tenth Edition,McGraw-Hill, 2002; and Pratt and Taylor, eds., Principles of DrugAction, Third Edition, Churchill Livingston, N.Y., 1990.

Administration of the BTK inhibitors of Formula (I) or Formula (II) orpharmaceutical composition of these compounds can be effected by anymethod that enables delivery of the compounds to the site of action.These methods include oral routes, intraduodenal routes, parenteralinjection (including intravenous, intraarterial, subcutaneous,intramuscular, intravascular, intraperitoneal or infusion), topical(e.g., transdermal application), rectal administration, via localdelivery by catheter or stent or through inhalation. The combination ofcompounds can also be administered intraadiposally or intrathecally.

Exemplary parenteral administration forms include solutions orsuspensions of active compound in sterile aqueous solutions, forexample, aqueous propylene glycol or dextrose solutions. Such dosageforms can be suitably buffered, if desired.

The invention also provides kits. The kits include the BTK inhibitors ofFormula (I) or Formula (II), either alone or in combination in suitablepackaging, and written material that can include instructions for use,discussion of clinical studies and listing of side effects. Such kitsmay also include information, such as scientific literature references,package insert materials, clinical trial results, and/or summaries ofthese and the like, which indicate or establish the activities and/oradvantages of the composition, and/or which describe dosing,administration, side effects, drug interactions, or other informationuseful to the health care provider. Such information may be based on theresults of various studies, for example, studies using experimentalanimals involving in vivo models and studies based on human clinicaltrials. The kit may further contain another active pharmaceuticalingredient. Suitable packaging and additional articles for use (e.g.,measuring cup for liquid preparations, foil wrapping to minimizeexposure to air, and the like) are known in the art and may be includedin the kit. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like. Kits may also, in selectedembodiments, be marketed directly to the consumer. In an embodiment, theinvention provides a kit comprising a BTK inhibitor of Formula (I) orFormula (II) for use in the treatment of CLL or SLL, hematologicalmalignancies, or any of the other cancers described herein.

Dosages and Dosing Regimens

The amounts of BTK inhibitors administered will be dependent on themammal being treated, the severity of the disorder or condition, therate of administration, the disposition of the compounds and thediscretion of the prescribing physician. However, an effective dosage isin the range of about 0.001 to about 100 mg per kg body weight per day,such as about 1 to about 35 mg/kg/day, in single or divided doses. For a70 kg human, this would amount to about 0.05 to 7 g/day, such as about0.05 to about 2.5 g/day. In some instances, dosage levels below thelower limit of the aforesaid range may be more than adequate, while inother cases still larger doses may be employed without causing anyharmful side effect—e.g., by dividing such larger doses into severalsmall doses for administration throughout the day.

In some embodiments, the BTK inhibitor of Formula (I) or Formula (II) isadministered in a single dose. Typically, such administration will be byinjection—e.g., intravenous injection, in order to introduce the agentsquickly. However, other routes may be used as appropriate. A single doseof a BTK inhibitor of Formula (I) or Formula (II) may also be used fortreatment of an acute condition.

In some embodiments, the BTK inhibitor of Formula (I) or Formula (II) isadministered in multiple doses. Dosing may be once, twice, three times,four times, five times, six times, or more than six times per day.Dosing may be once a month, once every two weeks, once a week, or onceevery other day. In other embodiments, a BTK inhibitor of Formula (I) orFormula (II) is administered about once per day to about 6 times perday. In some embodiments a BTK inhibitor of Formula (I) or Formula (II)is administered once daily, while in other embodiments a BTK inhibitorof Formula (I) or Formula (II) is administered twice daily, and in otherembodiments a BTK inhibitor of Formula (I) or Formula (II) isadministered three times daily.

Administration of the BTK inhibitor of Formula (I) or Formula (II) maycontinue as long as necessary. In some embodiments, the BTK inhibitor ofFormula (I) or Formula (II) is administered for more than 1, 2, 3, 4, 5,6, 7, 14, or 28 days. In some embodiments, the BTK inhibitor of Formula(I) or Formula (II) is administered for less than 28, 14, 7, 6, 5, 4, 3,2, or 1 day. In some embodiments, the BTK inhibitor of Formula (I) orFormula (II) is administered chronically on an ongoing basis—e.g., forthe treatment of chronic effects. In another embodiment theadministration of a BTK inhibitor of Formula (I) or Formula (II)continues for less than about 7 days. In yet another embodiment theadministration continues for more than about 6, 10, 14, 28 days, twomonths, six months, or one year. In some embodiments, continuous dosingis achieved and maintained as long as necessary.

In some embodiments, an effective dosage of a BTK inhibitor of Formula(I) or Formula (II) is in the range of about 1 mg to about 500 mg, about10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg,about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mgto about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202mg. In some embodiments, an effective dosage of a BTK inhibitor ofFormula (I) or Formula (II) is about 25 mg, about 50 mg, about 75 mg,about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg,about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg,about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg,about 475 mg, or about 500 mg. In some embodiments, an effective dosageof a BTK inhibitor of Formula (I) or Formula (II) is 25 mg, 50 mg, 75mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, or 500 mg.

In some embodiments, an effective dosage of a BTK inhibitor of Formula(I) or Formula (II) is in the range of about 0.01 mg/kg to about 4.3mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg toabout 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kgto about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg toabout 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kgto about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kgto about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kgto about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kgto about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg. In someembodiments, an effective dosage of a BTK inhibitor of Formula (I) orFormula (II) is about 0.35 mg/kg, about 0.7 mg/kg, about 1 mg/kg, about1.4 mg/kg, about 1.8 mg/kg, about 2.1 mg/kg, about 2.5 mg/kg, about 2.85mg/kg, about 3.2 mg/kg, or about 3.6 mg/kg.

In some embodiments, a BTK inhibitor of Formula (I) or Formula (II) isadminstered at a dosage of 10 to 400 mg BID, including a dosage of 25mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275mg, 300 mg, 325 mg, 350 mg, 375 mg, and 400 mg BID.

An effective amount of the combination of the BTK inhibitor of Formula(I) or Formula (II) may be administered in either single or multipledoses by any of the accepted modes of administration of agents havingsimilar utilities, including rectal, buccal, sublingual, intranasal andtransdermal routes, by intra-arterial injection, intravenously,intraperitoneally, parenterally, intramuscularly, subcutaneously,orally, topically, or as an inhalant.

Methods of Treating Hematological Malignancies, Cancers, and OtherDiseases

In an embodiment, the invention relates to a method of treating CLL in ahuman that comprises the step of administering to said human atherapeutically effective amount of a BTK inhibitor of Formula (II), ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof. In an embodiment, the invention relates to a methodof treating SLL in a human that comprises the step of administering tosaid human a therapeutically effective amount of a BTK inhibitor ofFormula (II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof. In an embodiment, the inventionrelates to a method of treating CLL in a human that comprises the stepof administering to said human a therapeutically effective amount of aBTK inhibitor of Formula (I), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof. In an embodiment,the invention relates to a method of treating SLL in a human thatcomprises the step of administering to said human a therapeuticallyeffective amount of a BTK inhibitor of Formula (I), or apharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof.

In an embodiment, the invention relates to a method of treating CLL in ahuman that comprises the step of administering to said human a BTKinhibitor of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof, in a dosingregimen selected from the group consisting of 100 mg QD, 175 mg QD, 250mg QD, 400 mg QD, and 100 mg BID. In an embodiment, the inventionrelates to a method of treating CLL in a human that comprises the stepof administering to said human a BTK inhibitor of Formula (I), or apharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof, in a dosing regimen selected from the groupconsisting of 100 mg QD, 175 mg QD, 250 mg QD, 400 mg QD, and 100 mgBID.

In an embodiment, the invention relates to a use of a composition ofFormula (II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof, in the manufacture of amedicament for treating CLL, wherein the treating comprises the step ofadministering one or more doses of Formula (II) or a pharmaceuticallyacceptable salt or ester, prodrug, cocrystal, solvate or hydratethereof. In an embodiment, the invention relates to a use of acomposition of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof, in themanufacture of a medicament for treating SLL, wherein the treatingcomprises the step of administering one or more doses of Formula (II) ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof. In an embodiment, the invention relates to a use ofa composition of Formula (I), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof, in themanufacture of a medicament for treating CLL, wherein the treatingcomprises the step of administering one or more doses of Formula (I) ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof. In an embodiment, the invention relates to a use ofa composition of Formula (I), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof, in themanufacture of a medicament for treating SLL, wherein the treatingcomprises the step of administering one or more doses of Formula (I) ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof.

In an embodiment, the invention relates to a method of treating CLL in amammal that comprises the step of administering to said mammal atherapeutically effective amount of a BTK inhibitor of Formula (II), ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof. In an embodiment, the invention relates to a methodof treating SLL in a mammal that comprises the step of administering tosaid mammal a therapeutically effective amount of a BTK inhibitor ofFormula (II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof. In an embodiment, the inventionrelates to a method of treating CLL in a mammal that comprises the stepof administering to said mammal a therapeutically effective amount of aBTK inhibitor of Formula (I), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof. In an embodiment,the invention relates to a method of treating SLL in a mammal thatcomprises the step of administering to said mammal a therapeuticallyeffective amount of a BTK inhibitor of Formula (I), or apharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof. In an embodiment, the mammal in any of the foregoingembodiments is selected from the group consisting of a human, a canine,a feline, or an equine. In an embodiment, the mammal in any of theforegoing embodiments is a companion animal.

In an embodiment, the invention relates to a method of treating asubtype of CLL in a human that comprises the step of administering tosaid mammal a therapeutically effective amount of a BTK inhibitor ofFormula (I) or Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof. A number ofsubtypes of CLL have been characterized. CLL is often classified forimmunoglobulin heavy-chain variable-region (IgV_(H)) mutational statusin leukemic cells. R. N. Damle, et al., Blood 1999, 94, 1840-47; T. J.Hamblin, et al., Blood 1999, 94, 1848-54. Patients with IgV_(H)mutations generally survive longer than patients without IgV_(H)mutations. ZAP70 expression (positive or negative) is also used tocharacterize CLL. L. Z. Rassenti, et al., N. Engl. J. Med. 2004, 351,893-901. The methylation of ZAP-70 at CpG3 is also used to characterizeCLL, for example by pyrosequencing. R. Claus, et al., J. Clin. Oncol.2012, 30, 2483-91; J. A. Woyach, et al., Blood 2014, 123, 1810-17. CLLis also classified by stage of disease under the Binet or Rai criteria.J. L. Binet, et al., Cancer 1977, 40, 855-64; K. R. Rai, T. Han,Hematol. Oncol. Clin. North Am. 1990, 4, 447-56. Other common mutations,such as 11p deletion, 13q deletion, and 17p deletion can be assessedusing well-known techniques such as fluorescence in situ hybridization(FISH). In an embodiment, the invention relates to a method of treatinga CLL in a human that comprises the step of administering to said mammala therapeutically effective amount of a BTK inhibitor of Formula (II),or a pharmaceutically acceptable salt or ester, prodrug, cocrystal,solvate or hydrate thereof, wherein the CLL is selected from the groupconsisting of IgV_(H) mutation negative CLL, ZAP-70 positive CLL, ZAP-70methylated at CpG3 CLL, CD38 positive CLL, chronic lymphocytic leukemiacharacterized by a 17p13.1 (17p) deletion, and CLL characterized by a11q22.3 (11q) deletion.

In an embodiment, the invention relates to a method of treating a CLL ina human that comprises the step of administering to said mammal atherapeutically effective amount of a BTK inhibitor of Formula (II), ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof, wherein the CLL has undergone a Richter'stransformation. Methods of assessing Richter's transformation, which isalso known as Richter's syndrome, are described in P. Jain and S.O'Brien, Oncology, 2012, 26, 1146-52. Richter's transformation is asubtype of CLL that is observed in 5-10% of patients. It involves thedevelopment of aggressive lymphoma from CLL and has a generally poorprognosis.

In an embodiment, the invention relates to a method of treating asubtype of CLL in a human, comprising the step of administering to saidmammal a therapeutically effective amount of a BTK inhibitor of Formula(II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof, wherein the subtype of CLL is asubtype of CLL that increases monocytes and NK cells in peripheral bloodwhen measured after a period of treatment with Formula (II) selectedfrom the group consisting of about 14 days, about 28 days, about 56days, about 1 month, about 2 months, about 3 months, about 6 months, andabout 1 year, and wherein the term “about” refers to a measurementinterval of +/−2 days.

In an embodiment, the invention relates to a method of treating chroniclymphocytic leukemia in a patient, wherein the chronic lymphocyticleukemia is chronic lymphocytic leukemia in a patient sensitive tolymphocytosis. In an embodiment, the invention relates to a method oftreating chronic lymphocytic leukemia in a patient, wherein the chroniclymphocytic leukemia is chronic lymphocytic leukemia in a patientexhibiting lymphocytosis caused by a disorder selected from the groupconsisting of a viral infection, a bacterial infection, a protozoalinfection, or a post-splenectomy state. In an embodiment, the viralinfection in any of the foregoing embodiments is selected from the groupconsisting of infectious mononucleosis, hepatitis, and cytomegalovirus.In an embodiment, the bacterial infection in any of the foregoingembodiments is selected from the group consisting of pertussis,tuberculosis, and brucellosis.

The methods described above may be used as first-line cancer therapy, orafter treatment with conventional chemotherapic active pharmaceuticalingredients, including cyclophosphamide, fludarabine, cyclophosphamideand fludarabine (FC chemotherapy), and chlorambucil. The methodsdescribed above may also be supplemented with immunotherapeuticmonoclonal antibodies such as the anti-CD52 monoclonal antibodyalemtuzumab. In an embodiment, the invention relates to a method oftreating CLL in a human that comprises the step of administering to saidhuman a therapeutically effective amount of a BTK inhibitor of Formula(II), or a pharmaceutically acceptable salt, ester, prodrug, cocrystal,solvate or hydrate thereof, and further comprises the step ofadministering to said human an active pharmaceutical ingredient selectedfrom the group consisting of cyclophosphamide, fludarabine,cyclophosphamide, chlorambucil, salts, esters, prodrugs, cocrystals,solvates, or hydrates thereof, and combinations thereof, andalemtuzumab, antigen-binding fragments, derivatives, conjugates,variants, and radioisotope-labeled complexes thereof.

In an embodiment, the invention relates to a method of treatinghematological malgnancies in a human comprising the step ofadministering to said human a therapeutically effective amount of a BTKinhibitor of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof. Hematologicalmalignancies include CLL and SLL, as well as other cancers of the blood,including B cell malignancies. In an embodiment, the invention relatesto a method of treating a hematological malignancy selected from thegroup consisting of non-Hodgkin's lymphoma (NHL), diffuse large B celllymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL),Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Waldenström's macroglobulinemia (WM), Burkitt's lymphoma, multiplemyeloma, or myelofibrosis in a human that comprises the step ofadministering a therapeutically effective amount of a BTK inhibitor ofFormula (II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating a NHLselected from the group consisting of indolent NHL and aggressive NHLcomprising the step of administering a therapeutically effective amountof a BTK inhibitor of Formula (II), or a pharmaceutically acceptablesalt or ester, prodrug, cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating a DLBCLselected from the group consisting of activated B-cell like diffuselarge B-cell lymphoma (DLBCL-ABC) and germinal center B-cell likediffuse large B-cell lymphoma (DLBCL-GCB), comprising the step ofadministering a therapeutically effective amount of a BTK inhibitor ofFormula (II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating an MCLselected from the group consisting of mantle zone MCL, nodular MCL,diffuse MCL, and blastoid MCL (also known as blastic variant MCL),comprising the step of administering a therapeutically effective amountof a BTK inhibitor of Formula (II), or a pharmaceutically acceptablesalt or ester, prodrug, cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating a B-ALLselected from the group consisting of early pre-B cell B-ALL, pre-B cellB-ALL, and mature B cell B-ALL (also known as Burkitt's leukemia),comprising the step of administering a therapeutically effective amountof a BTK inhibitor of Formula (II), or a pharmaceutically acceptablesalt or ester, prodrug, cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating aBurkitt's lymphoma selected from the group consisting of sporadicBurkitt's lymphoma, endemic Burkitt's lymphoma, and humanimmunodeficiency virus-associated Burkitt's lymphoma, comprising thestep of administering a therapeutically effective amount of a BTKinhibitor of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating amultiple myeloma selected from the group consisting of hyperdiploidmultiple myeloma and non-hyperdiploid multiple myeloma, comprising thestep of administering a therapeutically effective amount of a BTKinhibitor of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof.

In an embodiment, the invention relates to a method of treating amyelofibrosis selected from the group consisting of primarymyelofibrosis (also known as chronic idiopathic myelofibrosis) andmyelofibrosis secondary to polycythemia vera or essentialthrombocythaemia, comprising the step of administering a therapeuticallyeffective amount of a BTK inhibitor of Formula (II), or apharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof.

In an embodiment, the invention relates to a method of treating asubtype of a hematological malignancy in a human, comprising the step ofadministering to said human a therapeutically effective amount of a BTKinhibitor of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof, wherein thesubtype of a hematological malignancy is a subtype of a hematologicalmalignancy that increases monocytes and NK cells in peripheral bloodwhen measured after a period of treatment with Formula (II) selectedfrom the group consisting of about 14 days, about 28 days, about 56days, about 1 month, about 2 months, about 3 months, about 6 months, andabout 1 year, wherein the term “about” refers to a measurement intervalof +/−2 days, and wherein the hematological malignancy is selected fromthe group consisting of non-Hodgkin's lymphoma (NHL), diffuse large Bcell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma(MCL), Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Waldenström's macroglobulinemia (WM), Burkitt's lymphoma, multiplemyeloma, or myelofibrosis.

Methods of Treating Cancers in Patients Sensitive to Thrombosis

In selected embodiments, the invention provides a method of treating acancer in a human sensitive to platelet-mediated thrombosis, comprisingthe step of administering a therapeutically effective dose of a BTKinhibitor, or a pharmaceutically-acceptable salt, cocrystal, hydrate,solvate, or prodrug thereof. In an embodiment, the invention provides amethod of treating a cancer in a human sensitive to platelet-mediatedthrombosis, comprising the step of administering a therapeuticallyeffective dose of a BTK inhibitor, wherein the BTK inhibitor is Formula(II), or a pharmaceutically-acceptable salt, cocrystal, hydrate,solvate, or prodrug thereof. In an embodiment, the invention provides amethod of treating a cancer in a human sensitive to platelet-mediatedthrombosis, comprising the step of administering a therapeuticallyeffective dose of a BTK inhibitor, wherein the BTK inhibitor is Formula(II), or a pharmaceutically-acceptable salt, cocrystal, hydrate,solvate, or prodrug thereof, further comprising the step ofadministering a therapeutically effective dose of an anticoagulant orantiplatelet active pharmaceutical ingredient.

In selected embodiments, the BTK inhibitor of Formula (I) or Formula(II) and the anticoagulant or the antiplatelet active pharmaceuticalingredient are administered sequentially. In selected embodiments, theBTK inhibitor of Formula (I) or Formula (II) and the anticoagulant orthe antiplatelet active pharmaceutical ingredient are administeredconcomitantly. In selected embodiments, the BTK inhibitor of Formula (I)or Formula (II) is administered before the anticoagulant or theantiplatelet active pharmaceutical ingredient. In selected embodiments,the BTK inhibitor of Formula (I) or Formula (II) is administered afterthe anticoagulant or the antiplatelet active pharmaceutical ingredient.

In selected embodiments, the invention provides a method of treating acancer in a human sensitive to platelet-mediated thrombosis, comprisingthe step of administering a therapeutically effective dose of a BTKinhibitor, wherein the BTK inhibitor is Formula (II), and wherein thecancer is selected from the group consisting of CLL, SLL, NHL, DLBCL,FL, MCL, Hodgkin's lymphoma, B-ALL, WM, Burkitt's lymphoma, multiplemyeloma, or myelofibrosis that comprises the step of administering atherapeutically effective amount of a BTK inhibitor of Formula (II), ora pharmaceutically acceptable salt or ester, prodrug, cocrystal, solvateor hydrate thereof.

In selected embodiments, the invention provides a method of treating acancer in a human sensitive to platelet-mediated thrombosis, comprisingthe step of administering a therapeutically effective dose of a BTKinhibitor, wherein the BTK inhibitor is Formula (II), and wherein thecancer is selected from the group consisting of acute myeloid leukemia,squamous cell carcinoma including chronic myelocytic leukemia, bladdercancer, head and neck tumor, pancreatic ductal adenocarcinoma (PDA),pancreatic cancer, colon carcinoma, mammary carcinoma, breast cancer,fibrosarcoma, mesothelioma cancer, renal cell carcinoma, lung carcinoma,thyoma, prostate cancer, colorectal cancer, ovarian cancer, thymuscancer, brain cancer, squamous cell cancer, skin cancer, eye cancer,retinoblastoma, melanoma, intraocular melanoma, oral cavity andoropharyngeal cancers, gastric cancer, stomach cancer, cervical cancer,renal cancer, kidney cancer, liver cancer, ovarian cancer, prostatecancer, colorectal cancer, esophageal cancer, testicular cancer,gynecological cancer, thyroid cancer, acquired immune deficiencysyndrome (AIDS)-related cancers (e.g., lymphoma and Kaposi's sarcoma),viral-induced cancer, glioblastoma, esophageal tumors, hematologicalneoplasms, non-small-cell lung cancer, esophagus tumor, hepatitis Cvirus infection, hepatocellular carcinoma, metastatic colon cancer,multiple myeloma, ovary tumor, pancreas tumor, renal cell carcinoma,small-cell lung cancer, and stage IV melanoma.

In an embodiment, the invention provides a method of treating a cancerin a human sensitive to platelet-mediated thrombosis, comprising thestep of administering a therapeutically effective dose of a BTKinhibitor, wherein the BTK inhibitor is Formula (I) or Formula (II), ora pharmaceutically-acceptable salt, cocrystal, hydrate, solvate, orprodrug thereof, wherein the cancer is a hematological malignancy, andwherein the hematological malignancy is selected from the groupconsisting of chronic lymphocytic leukemia, B cell acute lymphoblasticleukemia, and non-Hodgkin's lymphoma.

In an embodiment, the invention provides a method of treating a cancerin a human sensitive to platelet-mediated thrombosis, comprising thestep of administering a therapeutically effective dose of a BTKinhibitor, wherein the BTK inhibitor is Formula (II), or apharmaceutically-acceptable salt, cocrystal, hydrate, solvate, orprodrug thereof, further comprising the step of administering atherapeutically effective dose of an anticoagulant or antiplateletactive pharmaceutical ingredient, wherein the cancer is a hematologicalmalignancy, and wherein the hematological malignancy is selected fromthe group consisting of chronic lymphocytic leukemia, B cell acutelymphoblastic leukemia, and non-Hodgkin's lymphoma.

Preferred anti-platelet and anticoagulant agents for use in the methodsof the present invention include, but are not limited to, cyclooxygenaseinhibitors (e.g., aspirin), adenosine diphosphate (ADP) receptorinhibitors (e.g., clopidogrel and ticlopidine), phosphodiesteraseinhibitors (e.g., cilostazol), glycoprotein IIb/IIIa inhibitors (e.g.,abciximab, eptifibatide, and tirofiban), adenosine reuptake inhibitors(e.g., dipyridamole), and acetylsalicylic acid (aspirin). Examples ofanti-platelet active pharmaceutical ingredients for use in the methodsof the present invention include acenocoumarol, anagrelide, anagrelidehydrochloride, abciximab, aloxiprin, antithrombin, apixaban, argatroban,aspirin, aspirin with extended-release dipyridamole, beraprost,betrixaban, bivalirudin, carbasalate calcium, cilostazol, clopidogrel,clopidogrel bisulfate, cloricromen, dabigatran etexilate, darexaban,dalteparin, dalteparin sodium, defibrotide, dicumarol, diphenadione,dipyridamole, ditazole, desirudin, edoxaban, enoxaparin, enoxaparinsodium, eptifibatide, fondaparinux, fondaparinux sodium, heparin,heparin sodium, heparin calcium, idraparinux, idraparinux sodium,iloprost, indobufen, lepirudin, low molecular weight heparin,melagatran, nadroparin, otamixaban, parnaparin, phenindione,phenprocoumon, prasugrel, picotamide, prostacyclin, ramatroban,reviparin, rivaroxaban, sulodexide, terutroban, terutroban sodium,ticagrelor, ticlopidine, ticlopidine hydrochloride, tinzaparin,tinzaparin sodium, tirofiban, tirofiban hydrochloride, treprostinil,treprostinil sodium, triflusal, vorapaxar, warfarin, warfarin sodium,ximelagatran, salts thereof, solvates thereof, hydrates thereof,cocrystals thereof, prodrugs thereof, and combinations thereof.

In an embodiment, the invention provides a method of treating a cancerin a human sensitive to platelet-mediated thrombosis, comprising thestep of administering a therapeutically effective dose of a BTKinhibitor, wherein the BTK inhibitor is Formula (II), or apharmaceutically-acceptable salt, cocrystal, hydrate, solvate, orprodrug thereof, further comprising the step of administering atherapeutically effective dose of an anticoagulant or antiplateletactive pharmaceutical ingredient, wherein the anticoagulant orantiplatelet active pharmaceutical ingredient is selected from the groupconsisting of acenocoumarol, anagrelide, anagrelide hydrochloride,abciximab, aloxiprin, antithrombin, apixaban, argatroban, aspirin,aspirin with extended-release dipyridamole, beraprost, betrixaban,bivalirudin, carbasalate calcium, cilostazol, clopidogrel, clopidogrelbisulfate, cloricromen, dabigatran etexilate, darexaban, dalteparin,dalteparin sodium, defibrotide, dicumarol, diphenadione, dipyridamole,ditazole, desirudin, edoxaban, enoxaparin, enoxaparin sodium,eptifibatide, fondaparinux, fondaparinux sodium, heparin, heparinsodium, heparin calcium, idraparinux, idraparinux sodium, iloprost,indobufen, lepirudin, low molecular weight heparin, melagatran,nadroparin, otamixaban, parnaparin, phenindione, phenprocoumon,prasugrel, picotamide, prostacyclin, ramatroban, reviparin, rivaroxaban,sulodexide, terutroban, terutroban sodium, ticagrelor, ticlopidine,ticlopidine hydrochloride, tinzaparin, tinzaparin sodium, tirofiban,tirofiban hydrochloride, treprostinil, treprostinil sodium, triflusal,vorapaxar, warfarin, warfarin sodium, ximelagatran, salts thereof,solvates thereof, hydrates thereof, cocrystals thereof, prodrugsthereof, and combinations thereof.

Combinations of BTK Inhibitors and Anti-CD20 Antibodies

The BTK inhibitors of Formula (I) and Formula (II) may also be safelyco-administered with immunotherapeutic antibodies such as the anti-CD20antibodies rituximab, obinutuzumab, ofatumumab, veltuzumab, tositumomab,and ibritumomab, and or antigen-binding fragments, derivatives,conjugates, variants, and radioisotope-labeled complexes thereof, whichmay be given alone or with conventional chemotherapeutic activepharmaceutical ingredients such as those described herein. The CD20antigen also called human B-lymphocyte-restricted differentiationantigen, Bp35, or B1) is found on the surface of normal “pre-B” andmature B lymphocytes, including malignant B lymphocytes. L. M. Nadler,et al., J. Clin. Invest. 1981, 67, 134-40; P. Stashenko, et al., J.Immunol. 1980, 139, 3260-85. The CD20 antigen is a glycosylated integralmembrane protein with a molecular weight of approximately 35 kD. T. F.Tedder, et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 208-12. CD20 isalso expressed on most B cell non-Hodgkin's lymphoma cells, but is notfound on hematopoietic stem cells, pro-B cells, normal plasma cells, orother normal tissues. Anti-CD20 antibodies are currently used astherapies for many hematological malignancies, including indolent NHL,aggressive NHL, and CLL/SLL. S. H. Lim, et. al., Haematologica 2010, 95,135-43; S. A. Beers, et. al., Sem. Hematol. 2010, 47, 107-14; C. Klein,et al., mAbs 2013, 5, 22-33.

In an embodiment, the invention relates to a method of treating ahematological malignancy in a human comprising the step of administeringto said human a BTK inhibitor of Formula (II), or a pharmaceuticallyacceptable salt or ester, prodrug, cocrystal, solvate or hydratethereof, and further comprising the step of administering an anti-CD20antibody, wherein the anti-CD20 antibody is a monoclonal antibody or anantigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. In an embodiment, the inventionrelates to a method of treating a hematological malignancy in a humancomprising the step of administering to said human a BTK inhibitor ofFormula (II), or a pharmaceutically acceptable salt or ester, prodrug,cocrystal, solvate or hydrate thereof, and further comprising the stepof administering an anti-CD20 antibody, wherein the anti-CD20 antibodyis an anti-CD20 monoclonal antibody or an antigen-binding fragment,derivative, conjugate, variant, or radioisotope-labeled complex thereof,and wherein the anti-CD20 antibody specifically binds to human CD20 witha K_(D) selected from the group consisting of 1×10⁻⁷ M or less, 5×10⁻⁸ Mor less, 1×10⁻⁸ M or less, and 5×10⁻⁹ M or less.

In an embodiment, the invention relates to a method of treating CLL orSLL in a human comprising the step of administering to said human a BTKinhibitor of Formula (II), or a pharmaceutically acceptable salt orester, prodrug, cocrystal, solvate or hydrate thereof, and furthercomprising the step of administering an anti-CD20 antibody, wherein theanti-CD20 antibody is a monoclonal antibody or an antigen-bindingfragment, derivative, conjugate, variant, or radioisotope-labeledcomplex thereof. In an embodiment, the invention relates to a method oftreating CLL or SLL in a human comprising the step of administering tosaid human a BTK inhibitor of Formula (II), or a pharmaceuticallyacceptable salt or ester, prodrug, cocrystal, solvate or hydratethereof, and further comprising the step of administering an anti-CD20antibody, wherein the anti-CD20 antibody is an anti-CD20 monoclonalantibody or an antigen-binding fragment, derivative, conjugate, variant,or radioisotope-labeled complex thereof, and wherein the anti-CD20antibody specifically binds to human CD20 with a K_(D) selected from thegroup consisting of 1×10⁻⁷ M or less, 5×10⁻⁸ M or less, 1×10⁻⁸ M orless, and 5×10⁻⁹ M or less.

In selected embodiments, the BTK inhibitor of Formula (I) or Formula(II) and the anti-CD20 monoclonal antibody are administeredsequentially. In selected embodiments, the BTK inhibitor of Formula (I)or Formula (II) and the anti-CD20 monoclonal antibody are administeredconcomitantly. In selected embodiments, the BTK inhibitor of Formula (I)or Formula (II) is administered before the anti-CD20 monoclonalantibody. In selected embodiments, the BTK inhibitor of Formula (I) orFormula (II) is administered after the anticoagulant or the antiplateletactive pharmaceutical ingredient. In selected embodiments, the BTKinhibitor of Formula (I) or Formula (II) and the anti-CD20 monoclonalantibody are administered over the same time period, and the BTKinhibitor administration continues after the anti-CD20 monoclonalantibody administration is completed.

In an embodiment, the anti-CD20 monoclonal antibody is rituximab, or anantigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. Rituximab is a chimericmurine-human monoclonal antibody directed against CD20, and itsstructure comprises an IgG1 kappa immunoglobulin containing murinelight- and heavy-chain variable region sequences and human constantregion sequences. Rituximab is composed of two heavy chains of 451 aminoacids and two light chains of 213 amino acids. The amino acid sequencefor the heavy chains of rituximab is set forth in SEQ ID NO:1. The aminoacid sequence for the light chains of rituximab is set forth in SEQ IDNO:2. Rituximab is commercially available, and its properties and use incancer and other diseases is described in more detail in W. Rastetter,et al., Ann. Rev. Med. 2004, 55, 477-503, and in G. L. Plosker and D. P.Figgett, Drugs, 2003, 63, 803-43. In an embodiment, the anti-CD20monoclonal antibody is an anti-CD20 biosimilar monoclonal antibodyapproved by drug regulatory authorities with reference to rituximab. Inan embodiment, the anti-CD20 monoclonal antibody has a heavy chainsequence identity of greater than 90% to SEQ ID NO:1. In an embodiment,the anti-CD20 monoclonal antibody has a light chain sequence identity ofgreater than 90% to SEQ ID NO:2. In an embodiment, the anti-CD20monoclonal antibody has a heavy chain sequence identity of greater than95% to SEQ ID NO:1. In an embodiment, the anti-CD20 monoclonal antibodyhas a light chain sequence identity of greater than 95% to SEQ ID NO:2.In an embodiment, the anti-CD20 monoclonal antibody has a heavy chainsequence identity of greater than 98% to SEQ ID NO:1. In an embodiment,the anti-CD20 monoclonal antibody has a light chain sequence identity ofgreater than 98% to SEQ ID NO:2. In an embodiment, the anti-CD20monoclonal antibody has a heavy chain sequence identity of greater than99% to SEQ ID NO:1. In an embodiment, the anti-CD20 monoclonal antibodyhas a light chain sequence identity of greater than 99% to SEQ ID NO:2.

In an embodiment, the anti-CD20 monoclonal antibody is obinutuzumab, oran antigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. Obinutuzumab is also known asafutuzumab or GA-101. Obinutuzumab is a humanized monoclonal antibodydirected against CD20. The amino acid sequence for the heavy chains ofobinutuzumab is set forth in SEQ ID NO:3. The amino acid sequence forthe light chains of obinutuzumab is set forth in SEQ ID NO:4.Obinutuzumab is commercially available, and its properties and use incancer and other diseases is described in more detail in T. Robak, Curr.Opin. Investig. Drugs 2009, 10, 588-96. In an embodiment, the anti-CD20monoclonal antibody is an anti-CD20 biosimilar monoclonal antibodyapproved by drug regulatory authorities with reference to obinutuzumab.In an embodiment, the anti-CD20 monoclonal antibody has a heavy chainsequence identity of greater than 90% to SEQ ID NO:3. In an embodiment,the anti-CD20 monoclonal antibody has a light chain sequence identity ofgreater than 90% to SEQ ID NO:4. In an embodiment, the anti-CD20monoclonal antibody has a heavy chain sequence identity of greater than95% to SEQ ID NO:3. In an embodiment, the anti-CD20 monoclonal antibodyhas a light chain sequence identity of greater than 95% to SEQ ID NO:4.In an embodiment, the anti-CD20 monoclonal antibody has a heavy chainsequence identity of greater than 98% to SEQ ID NO:3. In an embodiment,the anti-CD20 monoclonal antibody has a light chain sequence identity ofgreater than 98% to SEQ ID NO:4. In an embodiment, the anti-CD20monoclonal antibody has a heavy chain sequence identity of greater than99% to SEQ ID NO:3. In an embodiment, the anti-CD20 monoclonal antibodyhas a light chain sequence identity of greater than 99% to SEQ ID NO:4.In an embodiment, the anti-CD20 monoclonal antibody obinutuzumab is animmunoglobulin G1, anti-(human B-lymphocyte antigen CD20(membrane-spanning 4-domains subfamily A member 1, B-lymphocyte surfaceantigen B1, Leu-16 or Bp35)), humanized mouse monoclonal obinutuzumabdes-CH3107-K-γ1 heavy chain (222-219′)-disulfide with humanized mousemonoclonal obinutuzumab κ light chain dimer(228-228″:231-231″)-bisdisulfide antibody.

In an embodiment, the anti-CD20 monoclonal antibody is ofatumumab, or anantigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. Ofatumumab is described in B. D.Cheson, J. Clin. Oncol. 2010, 28, 3525-30. The crystal structure of theFab fragment of ofatumumab has been reported in Protein Data Bankreference 3GIZ and in J. Du, et al., Mol. Immunol. 2009, 46, 2419-2423.Ofatumumab is commercially available, and its preparation, properties,and use in cancer and other diseases is described in more detail in U.S.Pat. No. 8,529,202 B2, the disclosure of which is incorporated herein byreference. In an embodiment, the anti-CD20 monoclonal antibody is ananti-CD20 biosimilar monoclonal antibody approved by drug regulatoryauthorities with reference to ofatumumab. In an embodiment, theanti-CD20 monoclonal antibody has a variable heavy chain sequenceidentity of greater than 90% to SEQ ID NO:5. In an embodiment, theanti-CD20 monoclonal antibody has a variable light chain sequenceidentity of greater than 90% to SEQ ID NO:6. In an embodiment, theanti-CD20 monoclonal antibody has a variable heavy chain sequenceidentity of greater than 95% to SEQ ID NO:5. In an embodiment, theanti-CD20 monoclonal antibody has a variable light chain sequenceidentity of greater than 95% to SEQ ID NO:6. In an embodiment, theanti-CD20 monoclonal antibody has a variable heavy chain sequenceidentity of greater than 98% to SEQ ID NO:5. In an embodiment, theanti-CD20 monoclonal antibody has a variable light chain sequenceidentity of greater than 98% to SEQ ID NO:6. In an embodiment, theanti-CD20 monoclonal antibody has a variable heavy chain sequenceidentity of greater than 99% to SEQ ID NO:5. In an embodiment, theanti-CD20 monoclonal antibody has a variable light chain sequenceidentity of greater than 99% to SEQ ID NO:6. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment heavy chain sequenceidentity of greater than 90% to SEQ ID NO:7. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment light chain sequenceidentity of greater than 90% to SEQ ID NO:8. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment heavy chain sequenceidentity of greater than 95% to SEQ ID NO:7. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment light chain sequenceidentity of greater than 95% to SEQ ID NO:8. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment heavy chain sequenceidentity of greater than 98% to SEQ ID NO:7. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment light chain sequenceidentity of greater than 98% to SEQ ID NO:8. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment heavy chain sequenceidentity of greater than 99% to SEQ ID NO:7. In an embodiment, theanti-CD20 monoclonal antibody has a Fab fragment light chain sequenceidentity of greater than 99% to SEQ ID NO:8. In an embodiment, theanti-CD20 monoclonal antibody ofatumumab is an immunoglobulin G1,anti-(human B-lymphocyte antigen CD20 (membrane-spanning 4-domainssubfamily A member 1, B-lymphocyte surface antigen B1, Leu-16 or Bp35));human monoclonal ofatumumab-CD20 γ1 heavy chain (225-214′)-disulfidewith human monoclonal ofatumumab-CD20 κ light chain, dimer(231-231″:234-234″)-bisdisulfide antibody.

In an embodiment, the anti-CD20 monoclonal antibody is veltuzumab, or anantigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. Veltuzumab is also known as hA20.Veltuzumab is described in D. M. Goldenberg, et al., Leuk. Lymphoma2010, 51, 747-55. In an embodiment, the anti-CD20 monoclonal antibody isan anti-CD20 biosimilar monoclonal antibody approved by drug regulatoryauthorities with reference to veltuzumab. In an embodiment, theanti-CD20 monoclonal antibody has a heavy chain sequence identity ofgreater than 90% to SEQ ID NO:9. In an embodiment, the anti-CD20monoclonal antibody has a light chain sequence identity of greater than90% to SEQ ID NO:10. In an embodiment, the anti-CD20 monoclonal antibodyhas a heavy chain sequence identity of greater than 95% to SEQ ID NO:9.In an embodiment, the anti-CD20 monoclonal antibody has a light chainsequence identity of greater than 95% to SEQ ID NO:10. In an embodiment,the anti-CD20 monoclonal antibody has a heavy chain sequence identity ofgreater than 98% to SEQ ID NO:9. In an embodiment, the anti-CD20monoclonal antibody has a light chain sequence identity of greater than98% to SEQ ID NO:10. In an embodiment, the anti-CD20 monoclonal antibodyhas a heavy chain sequence identity of greater than 99% to SEQ ID NO:9.In an embodiment, the anti-CD20 monoclonal antibody has a light chainsequence identity of greater than 99% to SEQ ID NO:10. In an embodiment,the anti-CD20 monoclonal antibody ofatumumab is an immunoglobulin G1,anti-(human B-lymphocyte antigen CD20 (membrane-spanning 4-domainssubfamily A member 1, Leu-16, Bp35)); [218-arginine, 360-glutamic acid,362-methionine]humanized mouse monoclonal hA20 γ1 heavy chain(224-213′)-disulfide with humanized mouse monoclonal hA20 κ light chain(230-230″:233-233″)-bisdisulfide dimer

In an embodiment, the anti-CD20 monoclonal antibody is tositumomab, oran antigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. In an embodiment, the anti-CD20monoclonal antibody is ¹³¹I-labeled tositumomab. In an embodiment, theanti-CD20 monoclonal antibody is an anti-CD20 biosimilar monoclonalantibody approved by drug regulatory authorities with reference totositumomab. In an embodiment, the anti-CD20 monoclonal antibody has aheavy chain sequence identity of greater than 90% to SEQ ID NO:11. In anembodiment, the anti-CD20 monoclonal antibody has a light chain sequenceidentity of greater than 90% to SEQ ID NO:12. In an embodiment, theanti-CD20 monoclonal antibody has a heavy chain sequence identity ofgreater than 95% to SEQ ID NO:11. In an embodiment, the anti-CD20monoclonal antibody has a light chain sequence identity of greater than95% to SEQ ID NO:12. In an embodiment, the anti-CD20 monoclonal antibodyhas a heavy chain sequence identity of greater than 98% to SEQ ID NO:11.In an embodiment, the anti-CD20 monoclonal antibody has a light chainsequence identity of greater than 98% to SEQ ID NO:12. In an embodiment,the anti-CD20 monoclonal antibody has a heavy chain sequence identity ofgreater than 99% to SEQ ID NO:11. In an embodiment, the anti-CD20monoclonal antibody has a light chain sequence identity of greater than99% to SEQ ID NO:12.

In an embodiment, the anti-CD20 monoclonal antibody is ibritumomab, oran antigen-binding fragment, derivative, conjugate, variant, orradioisotope-labeled complex thereof. The active form of ibritumomabused in therapy is ibritumomab tiuxetan. When used with ibritumomab, thechelator tiuxetan (diethylene triamine pentaacetic acid) is complexedwith a radioactive isotope such as ⁹⁰Y or ¹¹¹In. In an embodiment, theanti-CD20 monoclonal antibody is ibritumomab tiuxetan, orradioisotope-labeled complex thereof. In an embodiment, the anti-CD20monoclonal antibody is an anti-CD20 biosimilar monoclonal antibodyapproved by drug regulatory authorities with reference to tositumomab.In an embodiment, the anti-CD20 monoclonal antibody has a heavy chainsequence identity of greater than 90% to SEQ ID NO:13. In an embodiment,the anti-CD20 monoclonal antibody has a light chain sequence identity ofgreater than 90% to SEQ ID NO:14. In an embodiment, the anti-CD20monoclonal antibody has a heavy chain sequence identity of greater than95% to SEQ ID NO:13. In an embodiment, the anti-CD20 monoclonal antibodyhas a light chain sequence identity of greater than 95% to SEQ ID NO:14.In an embodiment, the anti-CD20 monoclonal antibody has a heavy chainsequence identity of greater than 98% to SEQ ID NO:13. In an embodiment,the anti-CD20 monoclonal antibody has a light chain sequence identity ofgreater than 98% to SEQ ID NO:14. In an embodiment, the anti-CD20monoclonal antibody has a heavy chain sequence identity of greater than99% to SEQ ID NO:13. In an embodiment, the anti-CD20 monoclonal antibodyhas a light chain sequence identity of greater than 99% to SEQ ID NO:14.

In an embodiment, an anti-CD20 antibody selected from the groupconsisting of obinutuzumab, ofatumumab, veltuzumab, tositumomab, andibritumomab, and or antigen-binding fragments, derivatives, conjugates,variants, and radioisotope-labeled complexes thereof, is administered toa subject by infusion in a dose selected from the group consisting ofabout 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg,about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg,about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, andabout 2000 mg. In an embodiment, the anti-CD20 antibody is administeredweekly. In an embodiment, the anti-CD20 antibody is administeredmonthly. In an embodiment, the anti-CD20 antibody is administered at alower initial dose, which is escalated when administered at subsequentintervals administered monthly. For example, the first infusion candeliver 300 mg of anti-CD20 antibody, and subsequent weekly doses coulddeliver 2,000 mg of anti-CD20 antibody for eight weeks, followed bymonthly doses of 2,000 mg of anti-CD20 antibody. During any of theforegoing embodiments, the BTK inhibitors of Formula (I) or Formula (II)may be administered daily, twice daily, or at different intervals asdescribed above, at the dosages described above.

In an embodiment, the invention provides a kit comprising a compositioncomprising a BTK inhibitor of Formula (I) or Formula (II) and acomposition comprising an anti-CD20 antibody selected from the groupconsisting of rituximab, obinutuzumab, ofatumumab, veltuzumab,tositumomab, and ibritumomab, or an antigen-binding fragment,derivative, conjugate, variant, or radioisotope-labeled complex thereof,for use in the treatment of CLL or SLL, hematological malignancies, Bcell malignancies or, or any of the other diseases described herein. Thecompositions are typically both pharmaceutical compositions. The kit isfor use in co-administration of the anti-CD20 antibody and the BTKinhibitor, either simultaneously or separately, in the treatment of CLLor SLL, hematological malignancies, B cell malignancies, or any of theother diseases described herein.

Examples

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1 Preclinical Study of a Second Generation BTK Inhibitor for Usein CLL/SLL

The BTK inhibitor ibrutinib((1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one)is a first-generation BTK inhibitor. In clinical testing as amonotherapy in subjects with hematologic malignancies, ibrutinib wasgenerally well tolerated at dose levels through 840 mg (the highest dosetested). R. H. Advani, et al., J. Clin. Oncol. 2013, 31, 88-94; J. C.Byrd, et al., N. Engl. J. Med. 2013, 369, 32-42; M. L. Wang, et al., N.Engl. J. Med. 2013, 369, 507-16. No maximum tolerated dose (MTD) wasapparent within the tested dose range. Furthermore, subjects typicallyfound the drug tolerable over periods extending to >2 years. No subjecthad tumor lysis syndrome. No overt pattern of myelosuppression wasassociated with ibrutinib treatment. No drug-related reductions incirculating CD4⁺ T cells or serum immunoglobulins were noted. Adverseevents with an apparent relationship to study drug included diarrhea andrash.

In subjects with heavily pretreated non-Hodgkin lymphoma (NHL),ibrutinib showed substantial antitumor activity, inducing durableregressions of lymphadenopathy and splenomegaly in most subjects.Improvements in disease-associated anemia and thrombocytopenia wereobserved. The pattern of changes in subjects with CLL was notable.Single-agent ibrutinib caused rapid and substantial reductions in lymphnode size concomitant with a redistribution of malignant sites into theperipheral blood. An asymptomatic absolute lymphocyte count (ALC)increase was observed that was maximal during the first few months oftreatment and generally decreased thereafter but could be persistent insome subjects or could be seen repeatedly in subjects who hadinterruption and resumption of drug therapy.

Collectively, these data with ibrutinib support the potential benefitsof selective BTK inhibition in the treatment of subjects with relapsedlymphoid cancers. However, while highly potent in inhibiting BTK,ibrutinib has also shown in vitro activity against other kinases with acysteine in the same position as Cys481 in BTK to which the drugcovalently binds. For example, ibrutinib inhibits epidermal growthfactor receptor (EGFR), which may be the cause of ibrutinib-relateddiarrhea and rash. In addition, it is a substrate for both cytochromeP450 (CYP) enzymes 3A4/5 and 2D6, which increases the possibility ofdrug-drug interactions. These liabilities support the development ofalternative BTK inhibitors for use in the therapy of lymphoid cancer.

The preclinical selectivity and potency characteristics of thesecond-generation BTK inhibitor of Formula (II) were compared to thefirst-generation BTK inhibitor ibrutinib. In Table 1, a kinome screen(performed Life Technologies or based on literature data) is shown thatcompares these compounds.

TABLE 1 Kinome Screen for BTK Inhibitors (IC₅₀, nM) 3F-Cys KinaseFormula (II) Ibrutinib Btk 3.1 0.5 Tec 29 78 Bmx 39 0.80 Itk >1000 10.7Txk 291 2.0 EGFR >1000 5.6 ErbB2 912 9.4 ErbB4 13.2 2.7 Blk >1000 0.5JAK-3 >1000 16.1

The results shown in Table 1 are obtained from a 10 point biochemicalassay generated from 10 point concentration curves. The BTK inhibitor ofFormula (II) shows much greater selectivity for BTK compared to otherkinases than ibrutinib.

A comparison of the in vivo potency results for the BTK inhibitors ofFormula (II) and ibrutinib is shown in FIG. 1. CD86 and CD69 are cellsurface proteins that are BCR activation markers. To obtain the in vivopotency results, mice were gavaged at increasing drug concentration andsacrificed at one time point (3 h post-dose). BCR was stimulated withIgM and the expression of activation marker CD69 and CD86 are monitoredby flow cytometry and to determine EC₅₀ values.

Formula (II) is currently being evaluated in an ongoing study of caninespontaneous B-cell lymphoma. Six dogs have been treated with Formula(II) using 2.5 mg/kg once daily oral administration for an average of 22days (range 14 to 42 days). To date, partial remission (PR), perVeterinary Cooperative Oncology Group criteria for assessment ofresponse in peripheral nodal lymphoma, has been observed in 2 of 6 dogs.D. M. Vali, et al., Vet. Comp. Oncol. 8, 28-37 (2010). No drug-relatedadverse events have been reported to date in this study. These findingsare preliminary and similar to the clinical responses (i.e., 3 dogs withPR out of 8 dogs treated) observed with ibrutinib in dogs withspontaneous B-cell lymphoma. L. A. Honigberg, et al., Proc. Nat. Acad.Sci. USA, 107, 13075-13080 (2010).

In vitro and in vivo safety pharmacology studies with Formula (II) havedemonstrated a favorable nonclinical safety profile. When screened at 10μM in binding assays evaluating interactions with 80 known pharmacologictargets such as G-protein-coupled receptors, nuclear receptors,proteases, and ion channels, Formula (II) shows significant activityonly against the A3 adenosine receptor; follow-up dose-responseexperiments indicated a IC₅₀ of 2.7 μM, suggesting a low clinical riskof off-target effects. Formula (II) at 10 μM showed no inhibition of invitro EGFR phosphorylation in an A431 human epidermoid cancer cell linewhereas ibrutinib had an IC₅₀ of 66 nM. The in vitro effect of Formula(II) on human ether-à-go-go-related gene (hERG) channel activity wasinvestigated in vitro in human embryonic kidney cells stably transfectedwith hERG. Formula (II) inhibited hERG channel activity by 25% at 10 μM,suggesting a low clinical risk that Formula (II) would induce clinicalQT prolongation as predicted by this assay. Formula (II) was welltolerated in standard in vivo Good Laboratory Practices (GLP) studies ofpharmacologic safety. A functional observation battery in rats at dosesof through 300 mg/kg (the highest dose level) revealed no adverseeffects on neurobehavioral effects or body temperature at any doselevel. A study of respiratory function in rats also indicated notreatment-related adverse effects at doses through 300 mg/kg (thehighest dose level). In a cardiovascular function study in awaketelemeterized male beagle dogs, single doses of Formula (II) at doselevels through 30 mg/kg (the highest dose level) induced no meaningfulchanges in body temperature, cardiovascular, or electrocardiographic(ECG) (including QT interval) parameters. The results suggest thatFormula (II) is unlikely to cause serious off-target effects or adverseeffects on critical organ systems.

The drug-drug interaction potential of Formula (II) was also evaluated.In vitro experiments evaluating loss of parent drug as catalyzed by CYPsindicated that Formula (II) is metabolized by CYP3A4. In vitrometabolism studies using mouse, rat, dog, rabbit, monkey, and humanhepatocytes incubated with ¹⁴C-labeled Formula (II) indicated twomono-oxidized metabolites and a glutathione conjugate. No unique humanmetabolite was identified. Preliminary evaluations of metabolism in theplasma, bile, and urine of rats, dogs, and monkeys indicated metabolicprocesses of oxidation, glutathione binding, and hydrolysis. It wasshown that Formula (II) binds to glutathione but does not depleteglutathione in vitro. Nonclinical CYP interaction studies data indicatethat Formula (II) is very unlikely to cause clinical drug-druginteractions through alteration of the metabolism of drugs that aresubstrates for CYP enzymes.

Example 2 Clinical Study of a Second Generation BTK Inhibitor for Use inCLL/SLL

Clinical studies have shown that targeting the BCR signaling pathway byinhibiting BTK produces significant clinical benefit in patients withnon-Hodgkin's lymphoma (NHL). The second generation BTK inhibitor,Formula (II), achieves significant oral bioavailability and potency, andhas favorable preclinical characteristics, as described above. Thepurpose of this study is to evaluate the safety and efficacy of thesecond generation BTK inhibitor of Formula (II) in treating subjectswith chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma(SLL).

The design and conduct of this study is supported by an understanding ofthe history and current therapies for subjects with lymphoid cancers;knowledge of the activity and safety of a first-generation BTKinhibitor, ibrutinib, in subjects with hematologic cancers; and theavailable nonclinical information regarding Formula (II). The collectivedata support the following conclusions. BTK expression plays animportant role in the biology of lymphoid neoplasms, which representserious and life-threatening disorders with continuing unmet medicalneed. Clinical evaluation of Formula (II) as a potential treatment forthese disorders has sound scientific rationale based on observationsthat the compound selectively abrogates BTK activity and shows activityin nonclinical models of lymphoid cancers. These data are supported byclinical documentation that ibrutinib, a first-generation BTK inhibitor,is clinically active in these diseases. Ibrutinib clinical data andFormula (II) nonclinical safety pharmacology and toxicology studiessupport the safety of testing Formula (II) in subjects with B cellmalignancies.

The primary objectives of the clinical study are as follows: (1)establish the safety and the MTD of orally administered Formula (II) insubjects with CLL/SLL; (2) determine pharmacokinetics (PK) of orallyadministered Formula (II) and identification of its major metabolite(s);and (3) measure pharmacodynamic (PD) parameters including drug occupancyof BTK, the target enzyme, and effect on biologic markers of B cellfunction.

The secondary objective of the clinical study is to evaluate tumorresponses in patients treated with Formula (II).

This study is a multicenter, open-label, nonrandomized, sequentialgroup, dose escalation study. The following dose cohorts will beevaluated:

Cohort 1: 100 mg/day for 28 days (=1 cycle)

Cohort 2: 175 mg/day for 28 days (=1 cycle)

Cohort 3: 250 mg/day for 28 days (=1 cycle)

Cohort 4: 350 mg/day for 28 days (=1 cycle)

Cohort 5: 450 mg/day for 28 days (=1 cycle)

Cohort 6: To be determined amount in mg/day for 28 days (=1 cycle)

Each cohort will be enrolled sequentially with 6 subjects per cohort. If≦1 dose-limiting toxicity (DLT) is observed in the cohort during Cycle1, escalation to the next cohort will proceed. Subjects may be enrolledin the next cohort if 4 of the 6 subjects enrolled in the cohortcompleted Cycle 1 without experiencing a DLT, while the remaining 2subjects are completing evaluation. If ≧2 DLTs are observed during Cycle1, dosing at that dose and higher will be suspended and the MTD will beestablished as the previous cohort. The MTD is defined as the largestdaily dose for which fewer than 33% of the subjects experience a DLTduring Cycle 1. Dose escalation will end when either the MTD is achievedor at 3 dose levels above full BTK occupancy, whichever occurs first.Full BTK occupancy is defined as Formula (II) active-site occupancyof >80% (average of all subjects in cohort) at 24 hours postdose. Shouldescalation to Cohort 6 be necessary, the dose will be determined basedon the aggregate data from Cohorts 1 to 5, which includes safety,efficacy, and PK/PD results. The dose for Cohort 6 will not exceed 900mg/day.

Treatment with Formula (II) may be continued for >28 days until diseaseprogression or an unacceptable drug-related toxicity occurs. Subjectswith disease progression will be removed from the study. All subjectswho discontinue study drug will have a safety follow-up visit 30 (±7)days after the last dose of study drug unless they have started anothercancer therapy within that timeframe. Radiologic tumor assessment willbe done at screening and at the end of Cycle 2, Cycle 4, and Cycle 12and at investigator discretion. Confirmation of complete response (CR)will require bone marrow analysis and radiologic tumor assessment. Forsubjects who remain on study for >11 months, a mandatory bone marrowaspirate and biopsy is required in Cycle 12 concurrent with theradiologic tumor assessment.

All subjects will have standard hematology, chemistry, and urinalysissafety panels done at screening. This study also includes pancreaticfunction assessment (serum amylase and serum lipase) due to thepancreatic findings in the 28-day GLP rat toxicity study. Once dosingcommences, all subjects will be evaluated for safety once weekly for thefirst 4 weeks, every other week for Cycle 2, and monthly thereafter.Blood samples will be collected during the first week of treatment forPK/PD assessments. ECGs will be done at screening, and on Day 1-2, 8,15, 22, 28 of Cycle 1, Day 15 and 28 of Cycle 2, and monthly thereafterthrough Cycle 6. ECGs are done in triplicate for screening only.Thereafter, single ECG tests are done unless a repeat ECG testing isrequired.

Dose-limiting toxicity is defined as any of the following events (if notrelated to disease progression): (1) any Grade ≧3 non-hematologictoxicity (except alopecia) persisting despite receipt of a single courseof standard outpatient symptomatic therapy (e.g., Grade 3 diarrhea thatresponds to a single, therapeutic dose of Imodium® would not beconsidered a DLT); (2) grade ≧3 prolongation of the corrected QTinterval (QTc), as determined by a central ECG laboratory overread; (3)grade 4 neutropenia (absolute neutrophil count [ANC]<500/μL) lasting >7days after discontinuation of therapy without growth factors orlasting >5 days after discontinuation of therapy while on growth factors(i.e., Grade 4 neutropenia not lasting as long as specified will not beconsidered a DLT), (4) grade 4 thrombocytopenia (platelet count<20,000/μL) lasting >7 days after discontinuation of therapy orrequiring transfusion (i.e., Grade 4 thrombocytopenia not lasting aslong as specified will not be considered a DLT), and (5) dosing delaydue to toxicity for >7 consecutive days.

The efficacy parameters for the study include overall response rate,duration of response, and progression-free survival (PFS). The safetyparameters for the study include DLTs and MTD, frequency, severity, andattribution of adverse events (AEs) based on the Common TerminologyCriteria for Adverse Events (CTCAE v4.03) for non-hematologic AEs. M.Hallek, et al., Blood 2008, 111, 5446-5456.

The schedule of assessments is as follows, with all days stated in thefollowing meaning the given day or +/−2 days from the given day. Aphysical examination, including vital signs and weight, are performed atscreening, during cycle 1 at 1, 8, 15, 22, and 28 days, during cycle 2at 15 and 28 days, during cycles 3 to 24 at 28 days, and at follow up(after the last dose). The screening physical examination includes, at aminimum, the general appearance of the subject, height (screening only)and weight, and examination of the skin, eyes, ears, nose, throat,lungs, heart, abdomen, extremities, musculoskeletal system, lymphaticsystem, and nervous system. Symptom-directed physical exams are donethereafter. Vital signs (blood pressure, pulse, respiratory rate, andtemperature) are assessed after the subject has rested in the sittingposition. Eastern Cooperative Oncology Group (ECOG) status is assessedat screening, during cycle 1 at 1, 8, 15, 22, and 28 days, during cycle2 at 15 and 28 days, during cycles 3 to 24 at 28 days, and at follow up,using the published ECOG performance status indications described in M.M. Oken, et al., Am. J. Clin. Oncol. 1982, 5, 649-655. ECG testing isperformed at screening, during cycle 1 at 1, 2, 8, 15, 22, and 28 days,during cycle 2 at 15 and 28 days, during cycles 3 to 24 at 28 days, andat follow up. The 12-lead ECG test will be done in triplicate (≧1 minuteapart) at screening. The calculated QTc average of the 3 ECGs must be<480 ms for eligibility. On cycle 1, day 1 and cycle 1, day 8, singleECGs are done predose and at 1, 2, 4, and 6 h postdose. The single ECGon Cycle 1 Day 2 is done predose. On cycle 1, day 15, day 22, and day28, a single ECG is done 2 hours post-dose. Starting with cycle 2, asingle ECG is done per visit. Subjects should be in supine position andresting for at least 10 minutes before study-related ECGs. Twoconsecutive machine-read QTc>500 ms or >60 ms above baseline requirecentral ECG review. Hematology, including complete blood count withdifferential and platelet and reticulocyte counts, is assessed atscreening, during cycle 1 at 1, 8, 15, 22, and 28 days, during cycle 2at 15 and 28 days, during cycles 3 to 24 at 28 days, and at follow up.Serum chemistry is assessed at screening, during cycle 1 at 1, 8, 15,22, and 28 days, during cycle 2 at 15 and 28 days, during cycles 3 to 24at 28 days, and at follow up. Serum chemistry includes albumin, alkalinephosphatase, ALT, AST, bicarbonate, blood urea nitrogen (BUN), calcium,chloride, creatinine, glucose, lactate dehydrogenase (LDH), magnesium,phosphate, potassium, sodium, total bilirubin, total protein, and uricacid. Cell counts and serum immunoglobulin are performed at screening,at cycle 2, day 28, and at every 6 months thereafter until last dose andinclude T/B/NK/monocyte cell counts (CD3, CD4, CD8, CD14, CD19, CD19,CD16/56, and others as needed) and serum immunoglobulin (IgG, IgM, IgA,and total immunoglobulin). Bone marrow aspirates are performed at cycle12. Pharmacodynamics samples are drawn during cycle 1 at 1, 2, and 8days, and at follow up. On days 1 and 8, pharmacodynamic samples aredrawn pre-dose and 4 hours (±10 minutes) post-dose, and on day 2,pharmacodynamic samples are drawn pre-dose. Pharmacokinetics samples aredrawn during cycle 1 at 1, 2, 8, 15, 22, and 28 days. Pharmacokineticsamples for Cycle 1 Day 1 are drawn pre-dose and at 0.5, 1, 2, 4, 6 and24 hours (before dose on Day 2) post-dose. Samples for Cycle 1 Day 8 aredrawn pre-dose and at 0.5, 1, 2, 4, and 6 hours post-dose. On Cycle 1Day 15, 22, and 28, a PK sample is drawn pre-dose and the second PKsample must be drawn before (up to 10 minutes before) the ECGacquisition, which is 2 hours postdose. Pretreatment radiologic tumorassessments are performed within 30 days before the first dose. Acomputed tomography (CT) scan (with contrast unless contraindicated) isrequired of the chest, abdomen, and pelvis. In addition, a positronemission tomography (PET) or PET/CT must done for subjects with SLL.Radiologic tumor assessments are mandatory at the end of Cycle 2 (−7days), Cycle 4 (−7 days), and Cycle 12 (−7 days). Otherwise, radiologictumor assessments are done at investigator discretion. A CT (withcontrast unless contraindicated) scan of the chest, abdomen, and pelvisis required for subjects with CLL. In addition, a PET/CT is required insubjects with SLL. Bone marrow and radiologic assessments are bothrequired for confirmation of a complete response (CR). Clinicalassessments of tumor response should be done at the end of Cycle 6 andevery 3 months thereafter. Molecular markers are measured at screening,and include interphase cytogenetics, stimulated karyotype, IgHVmutational status, Zap-70 methylation, and beta-2 microglobulin levels.Urinalysis is performed at screening, and includes pH, ketones, specificgravity, bilirubin, protein, blood, and glucose. Other assessments,including informed consent, eligibility, medical history, and pregnancytest are done at the time of screening.

The investigator rates the subject's response to treatment based onrecent guidelines for CLL, as given in M. Hallek, et al., Blood 2008,111, 5446-56, and for SLL, as given in B. D. Cheson, et al., J. Clin.Oncol. 2007, 25, 579-586. The response assessment criteria for CLL aresummarized in Table 2.

TABLE 2 Response Assessment Criteria for CLL. Re- Bone Marrow (if Nodes,Liver, sponse Peripheral Blood performed) and Spleen^(a) CR Lymphocytes< 4 × 10⁹/L Normocellular < Normal (e.g., ANC > 1.5 × 10⁹/L^(b) 30%lymphocytes no lymph Platelets > 100 × 10⁹/L^(b) No B-lymphoid nodes >1.5 Hemoglobin > 11.0 g/dL nodules cm) (untransfused)^(b) CRiLymphocytes < 4 × 10⁹/L Hypocellular < Normal (e.g., Persistent anemia,30% lymphocytes no lymph thrombocytopenia, or nodes > 1.5 neutropeniarelated to cm) drug toxicity PR Lymphocytes ≧ 50% Not assessed ≧50%decrease from baseline reduction ANC > 1.5 × 10⁹/L in lymphade- ornopathy^(c) and/ Platelets > 100 × 10⁹/L or or in spleen 50% improvementover or liver baseline^(b) enlargement or Hemoglobin > 11.0 g/dL or 50%improvement over baseline (untransfused)^(b) Abbreviations: ANC =absolute neutrophil count; CR = complete remission; CRi = CR withincomplete blood count recovery; PR = partial remission. ^(a)Computedtomography (CT) scan of abdomen, pelvis, and chest is required for thisevaluation ^(b)Without need for exogenous growth factors ^(c)In the sumproducts of ≦6 lymph nodes or in the largest diameter of the enlargedlymph node(s) detected before therapy and no increase in any lymph nodeor new enlarged lymph nodes

The response assessment criteria for SLL are summarized in Table 3.

TABLE 3 Response Assessment Criteria for SLL. Response Definition NodalMasses Spleen, Liver Bone Marrow CR Disappearance (a) FDG-avid or PETNot palpable, If infiltrate present of all evidence positive prior tonodules at screening, of disease therapy; mass of any disappearedinfiltrate cleared on size permitted if PET repeat biopsy; if negativeindeterminate by (b) Variably FDG-avid morphology, or PET negative;immunohisto- regression to normal chemistry should be size on CTnegative PR Regression of ≧50% decrease in SPD ≧50% decrease Irrelevantif measurable of up to 6 largest in SPD of positive prior to disease andno dominant masses; no nodules (for therapy; cell type new sitesincrease in size of other single nodule in should be specified nodesgreatest (a) FDG-avid or PET transverse positive prior to diameter); notherapy; ≧1 PET increase in size positive at previously of liver orinvolved site spleen (b) Variably FDG-avid or PET negative; regressionon CT SD Failure to (a) FDG-avid or PET attain CR/PR positive prior toor progressive therapy; PET positive disease at prior sites of disease,and no new sites on CT or PET (b) Variably FDG avid or PET negative; nochange in size of previous lesions on CT Abbreviations: CR = completeremission, CT = computed tomography, FDG = [¹⁸F]fluorodeoxyglucose, PET= positron-emission tomography, PR = partial remission, SD = stabledisease, SPD = sum of the product of the diameters.

The PK parameters of the study are as follows. The plasma PK of Formula(II) and a metabolite is characterized using noncompartmental analysis.The following PK parameters are calculated, whenever possible, fromplasma concentrations of Formula (II):

-   -   AUC_((0-t)): Area under the plasma concentration-time curve        calculated using linear trapezoidal summation from time 0 to        time t, where t is the time of the last measurable concentration        (Ct),    -   AUC₍₀₋₂₄₎: Area under the plasma concentration-time curve from 0        to 24 hours, calculated using linear trapezoidal summation,    -   AUC_((0-∞)): Area under the plasma concentration-time curve from        0 to infinity, calculated using the formula:        AUC_((0-∞))=AUC_((0-t))+Ct/λz, where λz is the apparent terminal        elimination rate constant,    -   C_(max): Maximum observed plasma concentration,    -   T_(max): Time of the maximum plasma concentration (obtained        without interpolation),    -   t_(1/2): Terminal elimination half-life (whenever possible),    -   λ_(z): Terminal elimination rate constant (whenever possible),    -   Cl/F: Oral clearance.

The PD parameters of the study are as follows. The occupancy of BTK byFormula (II) are measured in peripheral blood mononuclear cells (PBMCs)with the aid of a biotin-tagged Formula (II) analogue probe. The effectof Formula (II) on biologic markers of B cell function will also beevaluated.

The statistical analysis used in the study is as follows. No formalstatistical tests of hypotheses are performed. Descriptive statistics(including means, standard deviations, and medians for continuousvariables and proportions for discrete variables) are used to summarizedata as appropriate.

The following definitions are used for the safety and efficacy analysissets: Safety analysis set: All enrolled subjects who receive ≧1 dose ofstudy drug; Per-protocol (PP) analysis set: All enrolled subjects whoreceive ≧1 dose of study drug and with ≧1 tumor response assessmentafter treatment. The safety analysis set will be used for evaluating thesafety parameters in this study. The PP analysis sets will be analyzedfor efficacy parameters in this study.

No imputation of values for missing data is performed except for missingor partial start and end dates for adverse events and concomitantmedication will be imputed according to prespecified, conservativeimputation rules. Subjects lost to follow-up (or drop out) will beincluded in statistical analyses to the point of their last evaluation.

The safety endpoint analysis was performed as follows. Safety summarieswill include summaries in the form of tables and listings. The frequency(number and percentage) of treatment emergent adverse events will bereported in each treatment group by Medical Dictionary for RegulatoryActivities (MedDRA) System Organ Class and Preferred Term. Summarieswill also be presented by the severity of the adverse event and byrelationship to study drug. Laboratory shift tables containing countsand percentages will be prepared by treatment assignment, laboratoryparameter, and time. Summary tables will be prepared for each laboratoryparameter. Figures of changes in laboratory parameters over time will begenerated. Vital signs, ECGs, and physical exams will be tabulated andsummarized.

Additional analyses include summaries of subject demographics, baselinecharacteristics, compliance, and concurrent treatments. Concomitantmedications will be coded according to the World Health Organization(WHO) Drug Dictionary and tabulated.

The analysis of efficacy parameters was performed as follows. The pointestimate of the overall response rate will be calculated for the PPanalysis set. The corresponding 95% confidence interval also will bederived. The duration of overall response is measured from the timemeasurement criteria are met for CR or PR (whichever is first recorded)until the first date that recurrent or progressive disease isobjectively documented (taking as reference for progressive disease thesmallest measurements recorded since the treatment started).Kaplan-Meier methodology will be used to estimate event-free curves andcorresponding quantiles (including the median). Progression-freesurvival is measured from the time of first study drug administrationuntil the first date that recurrent or progressive disease isobjectively documented (taking as reference for progressive disease thesmallest measurements recorded since the treatment started).Kaplan-Meier methodology will be used to estimate the event-free curvesand corresponding quantiles (including the median).

The study scheme is a sequential cohort escalation. Each cohort consistsof six subjects. The sample size of the study is 24 to 36 subjects,depending on dose escalation into subsequent cohorts. Cohort 1 (N=6)consists of Formula (II), 100 mg QD for 28 days. Cohort 2 (N=6) consistsof Formula (II), 175 mg QD for 28 days. Cohort 3 (N=6) consists ofFormula (II), 250 mg QD for 28 days. Cohort 4 (N=6) consists of Formula(II), 350 mg QD for 28 days. Cohort 5 (N=6) consists of Formula (II),450 mg QD for 28 days. Cohort 6 (N=6) consists of Formula (II), at adose to be determined QD for 28 days. The dose level for Cohort 6 willbe determined based on the safety and efficacy of Cohorts 1 to 5, andwill not exceed 900 mg/day. Escalation will end with either the MTDcohort or three levels above full BTK occupancy, whichever is observedfirst. An additional arm of the study will explore 100 mg BID dosing.Treatment with oral Formula (II) may be continued for greater than 28days until disease progression or an unacceptable drug-related toxicityoccurs.

The inclusion criteria for the study are as follows: (1) men and women≧18 years of age with a confirmed diagnosis of CLL/SLL, which hasrelapsed after, or been refractory to, ≧2 previous treatments forCLL/SLL; however, subjects with 17p deletion are eligible if they haverelapsed after, or been refractory to, 1 prior treatment for CLL/SLL;(2) body weight ≧60 kg, (3) ECOG performance status of ≦2; (4) agreementto use contraception during the study and for 30 days after the lastdose of study drug if sexually active and able to bear children; (5)willing and able to participate in all required evaluations andprocedures in this study protocol including swallowing capsules withoutdifficulty; or (6) ability to understand the purpose and risks of thestudy and provide signed and dated informed consent and authorization touse protected health information (in accordance with national and localsubject privacy regulations).

The dosage form and strength of Formula (II) used in the clinical studyis a hard gelatin capsules prepared using standard pharmaceutical gradeexcipients (microcrystalline cellulose) and containing 25 mg of Formula(II) each. The color of the capsules is Swedish orange. The route ofadministration is oral (per os, or PO). The dose regimen is once dailyor twice daily, as defined by the cohort, on an empty stomach (definedas no food 2 hours before and 30 minutes after dosing).

The baseline characteristics for the patients enrolled in the clinicalstudy are given in Table 4.

TABLE 4 Relapsed/refractory CLL baseline characteristics. CharacteristicCLL (N = 44) Patient Demographics Age (years), median (range)    62(45-84) Sex, men (%) 33 (75) Prior therapies, median (range), n    3(1-10) ≧3 prior therapies, n (%) 26 (59) Clinical Details ECOGperformance status ≧ 1 (%) 28 (63) Rai stage III/IV 16 (36) Bulkydisease ≧ 5 cm, n (%) 15 (34) Cytopenia at baseline 33 (75) CytogenicStatus Chromosome 11q22.3 deletion (Del 11q), n (%) 18 (41) Chromosome17p13.1 (Del 17p), n (%) 19 (34) IgV_(H) status (unmutated), n (%) 28(64)

The results of the clinical study in relapsed/refractory CLL patientsare summarized in Table 5.

TABLE 5 Activity of Formula (II) in relapsed/refractory CLL. (PR =partial response; PR + L = partial response with lymphocytosis; SD =stable disease; PD = progressive disease.) n (%) 175 mg 250 mg 100 mg400 mg All Cohorts 100 mg QD QD QD BID QD (N = 31)^(†) (N = 8) (N = 8)(N = 7) (N = 3) (N = 5) PR 22 (71) 7 (88) 5 (63) 5 (71) 3 (100) 2 (40)PR + L 7 (23) 0 (0) 3 (37) 2 (29) 0 (0) 2 (40) SD 2 (6) 1 (12) 0 (0) 0(0) 0 (0) 1 (20) PD 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Median (range)Cycles 7.3 (3.0-10.8) 10.0 (9.0-10.8) 8.6 (3.0-8.8) 7.0 (7.0-7.3) 5.2(4.7-5.5) 5.0 (4.8-5.5)

FIG. 2 shows the median % change in ALC and SPD from baseline in theclinical study of Formula (II), plotted in comparison to the resultsreported for ibrutinib in FIG. 1A of J. C. Byrd, et al., N. Engl. J.Med. 2013, 369, 32-42. The results show that Formula (II) leads to amore rapid patient response in CLL than corresponding treatment withibrutinib. This effect is illustrated, for example, by the median %change in SPD, which achieved the same status in the present study at 7months of treatment with Formula (II) as compared to 18 months foribrutinib. The % change in SPD observed in the different cohorts (i.e.by dose and dosing regimen) is shown in FIG. 3, and in all cases showssignificant responses.

A Kaplan-Meier curve showing PFS from the clinical CLL study of Formula(II) is shown in FIG. 4. A comparison of survival curves was performedusing the Log-Rank (Mantle-Cox) test, with a p-value of 0.0206indicating that the survival curves are different. The number ofpatients at risk is shown in FIG. 5. Both FIG. 4 and FIG. 5 show theresults for Formula (II) in comparison to the results reported foribrutinib in J. C. Byrd, et al., N. Engl. J. Med. 2013, 369, 32-42. Animprovement in survival and a reduction in risk are observed in CLLpatients treated with Formula (II) in comparison to patients treatedwith ibrutinib.

Based on the data and comparisons shown in FIG. 2 to FIG. 5, the CLLstudy with Formula (II) showed that the efficacy of Formula (II) wassurprisingly superior to that of ibrutinib.

In the literature study of ibrutinib, increased disease progression wasassociated with patients with high-risk cytogenetic lesions (17p13.1deletion or 11q22.3 deletion), as shown in FIG. 3A in J. C. Byrd, etal., N. Engl. J. Med. 2013, 369, 32-42, which shows ibrutinib PFSincluding PFS broken down by genetic abnormality. The 17p and 11qdeletions are validated high-risk characteristics of CLL, and the 17pdeletion is the highest risk. In FIG. 6, the PFS is shown for Formula(II) in patients with the 17p deletion in comparison to the resultsobtained for ibrutinib in J. C. Byrd, et al., N. Engl. J. Med. 2013,369, 32-42. A p-value of 0.0696 was obtained. In FIG. 7, the number ofpatients at risk with the 17p deletion is compared. To date, no 17ppatients have progressed on Formula (II).

The adverse events observed in the clinical study in relapsed/refractoryCLL are given in Table 6. No DLTs were observed. The MTD was notreached. No treatment-related serious adverse events (SAEs) wereobserved. No prophylactic antivirals or antibiotics were needed.

TABLE 6 Treatment-related adverse events reported in the clinical studyof Formula (II) in relapsed/refractory CLL. (Reported in ≧5% ofpatients.) Adverse Events (Treatment- Related), n (%) Grade All (N = 44)Headache ½ 7 (16) Increased tendency to bruise 1 6 (14) Diarrhea 1 4(9)  Petechiae 1 3 (7) 

The clinical study of Formula (II) thus showed other unexpectedlysuperior results compared to ibrutinib therapy. A lack of lymphocytosiswas observed in the study. Furthermore, only grade 1 AEs were observed,and these AEs were attributable to the high BTK selectivity of Formula(II).

BTK target occupancy was measured for relapsed/refractory CLL patientswith the results shown in FIG. 8. For 200 mg QD dosing of the BTKinhibitor of Formula (II), approximately 94%-99% BTK occupancy wasobserved, with superior 24 hour coverage and less inter-patientvariability also observed. For 420 mg and 840 mg QD of the BTK inhibitoribrutinib, 80%-90% BTK occupancy was observed, with more inter-patientvariability and capped occupancy. These results indicate that the BTKinhibitor of Formula (II) achieves superior BTK occupancy in CLLpatients than ibrutinib.

The effects of Formula (II) on cell subset percentages were alsoevaluated using flow cytometry analysis of peripheral blood, with theresults shown in FIG. 9, FIG. 10, FIG. 11, FIG. 12, FIG. 13, and FIG.14. PBMC samples from CLL patient samples drawn prior to (predose) andafter 28 days of dosing with Formula (II) were compared for potentialchanges in cell subsets. PBMCs were stained with monoclonal antibodiesconjugated to fluorescent tags (flourochromes) to identify cell subsetsvia flow cytometry. Non-viable cells were excluded from the analysisusing the dye 7-aminoactinomycin D (7-AAD). To produce the metric ofpercent change, the following steps were taken. First, each cell subsetwas defined by hierarchical flow cytometry gating. Then, the change infrequency (between day 1 and day 28) was calculated for each cellsubset. MDSC subsets were measured as a % of all myeloid cells. T cellsubsets were measured as a % of all CD3⁺ cells, and NK cells weremeasured as a % of all live CD45⁺ cells. In FIG. 9 and FIG. 10, theresults show the % change in MDSC (monocytic) level over 28 days versus% ALC change at cycle 1 day 28 (C1D28) and at cycle 2 day 28 (C2D28). Acycle is 28 days. A trend is observed wherein patients with decreasingALC % had increasing MDSC (monocytic) %. This may include patients whohad quickly resolving lymphocytosis and those with no initiallymphocytosis. This provides evidence that treatment with Formula (II)mobilizes MDSCs and thus affects the CLL tumor microenvironment inmarrow and lymph nodes, which is an unexpected indication of superiorefficacy. In FIG. 11 and FIG. 12, the results show the % change in NKcell level over 28 days versus % ALC change, measured at C1D28 or C2D28,and similar trends are observed wherein patients with decreasing ALC %had increasing NK cell %. This may include patients who had quicklyresolving lymphocytosis and those having no initial lymphocytosis. Theeffects in FIG. 9 to FIG. 12 are observed in multiple cohorts, at dosesincluding 100 mg BID, 200 mg QD, and 400 mg QD. In FIG. 13 and FIG. 14,the effects on NK cells and MDSC cells are compared to a number of othermarkers versus % change in ALC at C1D28 and C2D28. These other markersinclude CD4+ T cells, CD8+ T cells, CD4+/CD8+ T cell ratio, NK-T cells,PD-1+CD4+ T cells, and PD-1+CD8+ T cells. The effects on NK cells andMDSC cells are observed to be much more pronounced than on any of theseother markers.

These results suggest that after Formula (II) administration, the CLLmicroenvironment undergoes a change wherein NK cells and monocytic MDSCsubsets increase in frequency in the peripheral blood in patients withfalling ALC counts, an important clinical parameter in CLL. The NK cellincrease may reflect an overall increase in cytolytic activity againstB-CLL resulting in the ALC % to drop. The increase in MDSC % in theblood may be due to a movement of these cells out of the lymph nodes,spleen, and bone marrow, which are all possible sites of CLLproliferation. Fewer MDSCs at the CLL proliferation centers would likelyresult in a reduced immunosuppressive microenvironment leading to anincrease in cell-mediated immunity against the tumor, decreased tumorproliferation, and eventually lower ALC % in the circulation.

Overall, Formula (II) shows superior efficacy as measured by ALC thanfirst generation BTK inhibitors such as ibrutinib, or PI3K-δ inhibitorssuch as idelalisib. Formula (II) has better target occupancy and betterpharmacokinetic and metabolic parameters than ibrutinib, leading toimproved B cell apoptosis. Furthermore, unlike treatment with ibrutiniband PI3K-δ inhibitors, treatment with Formula (II) does not affect NKcell function. Finally, treatment with Formula (II) leads to a CLL tumormicroenvironmental effect by excluding MDSC cells from the marrow andlymph nodes and reducing their number.

Example 3 Effects of BTK Inhibitors on Thrombosis

Clinical studies have shown that targeting the BCR signaling pathway byinhibiting BTK produces significant clinical benefit (J. C. Byrd, etal., N. Engl. J. Med. 2013, 369, 32-42; M. L. Wang, et al., N. Engl. J.Med. 2013, 369, 507-16). However, in these studies, bleeding has beenreported in up to 50% of ibrutinib-treated patients. Most bleedingevents were of grade 1-2 (spontaneous bruising or petechiae) but, in 5%of patients, they were of grade 3 or higher after trauma. These resultsare reflected in the prescribing information for ibrutinib, wherebleeding events of any grade, including bruising and petechiae, werereported in approximately half of patients treated with ibrutinib(IMBRUVICA package insert and prescribing information, revised July2014, U.S. Food and Drug Administration).

Constitutive or aberrant activation of the BCR signaling cascade hasbeen implicated in the propagation and maintenance of a variety of Bcell malignancies. Small molecule inhibitors of BTK, a protein early inthis cascade and specifically expressed in B cells, have emerged as anew class of targeted agents. There are several BTK inhibitors,including CC-292 and ibrutinib (PCI-32765), in clinical development.CC-292 refers to(N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide,or a pharmaceutically acceptable salt thereof, including a hydrochloridesalt or besylate salt thereof. Importantly, early stage clinical trialshave found ibrutinib to be particularly active in chronic lymphocyticleukemia (CLL) and mantle cell lymphoma (MCL), suggesting that thisclass of inhibitors may play a significant role in various types ofcancers (Aalipour and Advani, Br. J. Haematol. 2013, 163, 436-43).However, their effects are not limited to leukemia or lymphomas asplatelets also rely on the Tec kinases family members BTK and Tec forsignal transduction in response to various thrombogenic stimuli (Oda, etal., Blood 2000, 95(5), 1663-70; Atkinson, et al. Blood 2003, 102(10),3592-99). In fact, both Tec and BTK play an important role in theregulation of phospholipase Cγ2 (PLCγ2) downstream of GPVI in humanplatelets. In addition, BTK is activated and undergoes tyrosinephosphorylation upon challenge of the platelet thrombin receptor, whichrequires the engagement of αIIbβ3 integrin and PI3K activity (Laffargue,et al., FEBS Lett. 1999, 443(1), 66-70). It has also been implicated inGPIbα-dependent thrombus stability at sites of vascular injury (Liu, etal., Blood 2006, 108(8), 2596-603). Thus, BTK and Tec are involved inseveral processes important in supporting the formation of a stablehemostatic plug, which is critical for preventing significant blood lossin response to vascular injury. Hence, the effects of the BTK inhibitorof Formula (II) and ibrutinib were evaluated on human platelet-mediatedthrombosis by utilizing the in vivo human thrombus formation in the VWFHA1 mice model described in Chen, et al. Nat. Biotechnol. 2008, 26(1),114-19.

Administration of anesthesia, insertion of venous and arterialcatheters, fluorescent labeling and administration of human platelets(5×10⁸/ml), and surgical preparation of the cremaster muscle in micehave been previously described (Chen, et al., Nat Biotechnol. 2008,26(1), 114-19). Injury to the vessel wall of arterioles (˜40-65 mmdiameter) was performed using a pulsed nitrogen dye laser (440 nm,Photonic Instruments) applied through a 20× water-immersion Olympusobjective (LUMPlanFl, 0.5 numerical aperture (NA)) of a Zeiss Axiotechvario microscope. Human platelet and wall interactions were visualizedby fluorescence microscopy using a system equipped with a YokogawaCSU-22 spinning disk confocal scanner, iXON EM camera, and 488 nm and561 nm laser lines to detect BCECF-labeled and rhodamine-labeledplatelets, respectively (Revolution XD, Andor Technology). The extent ofthrombus formation was assessed for 2 minutes after injury and the area(μm²) of coverage determined (Image IQ, Andor Technology). For theFormula (II), CC-292, ibrutinib inhibition studies, the BTK inhibitorswere added to purified human platelets for 30 minutes beforeadministration.

The in vivo thrombus effects of the BTK inhibitors, Formula (II),CC-292, and ibrutinib, were evaluated on human platelet-mediatedthrombosis by utilizing the in vivo human thrombus formation in the VWFHA1 mice model, which has been previously described (Chen, et al., NatBiotechnol. 2008, 26(1), 114-19). Purified human platelets werepreincubated with various concentrations of the BTK inhibitors (0.1 μM,0.5 μM, or 1 μM) or DMSO and then administered to VWF HA1 mice, followedby laser-induced thrombus formation. The BTK inhibitor-treated humanplatelets were fluorescently labeled and infused continuously through acatheter inserted into the femoral artery. Their behavior in response tolaser-induced vascular injury was monitored in real time usingtwo-channel confocal intravital microscopy (Furie and Furie, J. Clin.Invest. 2005, 115(12), 2255-62). Upon induction of arteriole injuryuntreated platelets rapidly formed thrombi with an average thrombus sizeof 6,450±292 mm² (mean±s.e.m.), as shown in FIGS. 15, 16, and 17.Similarly, Formula (II) (1 μM) treated platelets formed a slightlysmaller but not significantly different thrombi with an average thrombussize of 5733±393 mm² (mean±s.e.m.). In contrast, a dramatic reduction inthrombus size occurred in platelets pretreated with 1 μM of ibrutinib,2600±246 mm² (mean±s.e.m.), resulting in a reduction in maximal thrombussize by approximately 61% compared with control (P>0.001) (FIGS. 15 and17). Similar results were obtained with platelets pretreated with 500 nMof Formula (II) or ibrutinib: thrombus size of 5946±283 mm², and2710±325 mm² respectively. These initial results may provide somemechanic background and explanation on the reported 44% bleeding relatedadverse event rates in the Phase III RESONATE™ study comparing ibrutinibwith ofatumumab. The results obtained for CC-292 were similar to thatfor ibrutinib, as shown in FIGS. 15, 16, and 17. The effect of the BTKinhibitor concentration is shown in FIG. 18. These results demonstratethe surprising advantage of the BTK inhibitor of Formula (II), whichdoes not interfere with thrombus formation, while the BTK inhibitorsCC-292 and ibrutinib interfere with thrombus formation.

The objective of this study was to evaluate in vivo thrombus formationin the presence of BTK inhibitors. In vivo testing of novel antiplateletagents requires informative biomarkers. By utilizing a genetic modifiedmouse von Willebrand factor (VWFR1326H) model that supports human butnot mouse platelet-mediated thrombosis, we evaluated the effects ofFormula (II), CC-292, and ibrutinib on thrombus formation. These resultsshow that Formula (II) had no significant effect on humanplatelet-mediated thrombus formation while ibrutinib was able to limitthis process, resulting in a reduction in maximal thrombus size by 61%compared with control. CC-292 showed an effect similar to ibrutinib.These results, which show reduced thrombus formation for ibrutinib atphysiologically relevant concentrations, may provide some mechanisticbackground for the Grade ≧3 bleeding events (eg, subdural hematoma,gastrointestinal bleeding, hematuria and postprocedural hemorrhage) thathave been reported in ≦6% of patients treated with ibrutinib.

GPVI platelet aggregation was measured for Formula (II) and ibrutinib.Blood was obtained from untreated humans, and platelets were purifiedfrom plasma-rich protein by centrifugation. Cells were resuspended to afinal concentration of 350,000/μL in buffer containing 145 mmol/L NaCl,10 mmol/L HEPES, 0.5 mmol/L Na₂HPO₄, 5 mmol/L KCl, 2 mmol/L MgCl₂, 1mmol/L CaCl₂, and 0.1% glucose, at pH 7.4. Stock solutions of Convulxin(CVX) GPVI were prepared on the day of experimentation and added toplatelet suspensions 5 minutes (37° C., 1200 rpm) before the inductionof aggregation. Aggregation was assessed with a ChronologLumi-Aggregometer (model 540 VS; Chronolog, Havertown, Pa.) andpermitted to proceed for 6 minutes after the addition of agonist. Theresults are reported as maximum percent change in light transmittancefrom baseline with platelet buffer used as a reference. The results areshown in FIG. 19.

In FIG. 20, the results of CVX-induced (250 ng/mL) human plateletaggregation results before and 15 minutes after administration of theBTK inhibitors to 6 healthy individuals are shown.

The results depicted in FIG. 19 and FIG. 20 indicate that the BTKinhibitor ibrutinib significantly inhibits GPVI platelet aggregation,while the BTK inhibitor of Formula (II) does not, further illustratingthe surprising benefits of the latter compound.

Example 4 Effects of BTK Inhibition on Antibody-Dependent NK CellMediated Cytotoxicity

Rituximab-combination chemotherapy is today's standard of care in CD20⁺B-cell malignancies. Previous studies investigated and determined thatibrutinib antagonizes rituximab antibody-dependent cell mediatedcytotoxicity (ADCC) mediated by NK cells. This may be due to ibrutinib'ssecondary irreversible binding to interleukin-2 inducible tyrosinekinase (ITK) which is required for FcR-stimulated NK cell functionincluding calcium mobilization, granule release, and overall ADCC. H. E.Kohrt, et al., Blood 2014, 123, 1957-60.

In this example, the effects of Formula (II) and ibrutinib on NK cellfunction were evaluated in primary NK cells from healthy volunteers andCLL patients. The activation of NK cells co-cultured withantibody-coated target cells was strongly inhibited by ibrutinib. Thesecretion of IFN-γ was reduced by 48% (p=0.018) and 72% (p=0.002) incultures treated with ibrutinib at 0.1 and 1.0 μM respectively and NKcell degranulation was significantly (p=0.002) reduced, compared withcontrol cultures. Formula (II) treatment at 1 μM, a clinically relevantconcentration, did not inhibit IFN-γ or NK cell degranulation.Rituximab-mediated ADCC was evaluated in NK cells from healthyvolunteers as well as assays of NK cells from CLL patients targetingautologous CLL cells. In both cases, ADCC was not inhibited by Formula(II) treatment at 1 μM. In contrast, addition of ibrutinib to the ADCCassays strongly inhibited the rituximab-mediated cytotoxicity of targetcells, and no increase over natural cytotoxicity was observed at anyrituximab concentration. This result indicates that the combination ofrituximab and Formula (II) provides an unexpected benefit in thetreatment of CLL.

BTK is a non-receptor enzyme in the Tec kinase family that is expressedamong cells of hematopoietic origin, including B cells, myeloid cells,mast cells and platelets, where it regulates multiple cellular processesincluding proliferation, differentiation, apoptosis, and cell migration.W. N. Khan, Immunol Res. 2001, 23, 147-56; A. J. Mohamed, et al.,Immunol Rev. 2009, 228, 58-73; J. M. Bradshaw, Cell Signal. 2010, 22,1175-84. Functional null mutations of BTK in humans cause the inheriteddisease, X linked agammaglobulinemia, which is characterized by a lackof mature peripheral B cells. M. Vihinen, et al., Front Biosci. 2000, 5,D917-28. Conversely, BTK activation is implicated in the pathogenesis ofseveral B-cell malignancies. S. E. Herman, et al., Blood 2011, 117,6287-96; L. P. Kil, et al., Am. J. Blood Res. 2013, 3, 71-83; Y. T. Tai,et al., Blood 2012, 120, 1877-87; J. J. Buggy, L. Elias, Int. Rev.Immunol. 2012, 31, 119-32 (Erratum in: Int. Rev. Immunol. 2012, 31,428). In addition, BTK-dependent activation of mast cells and otherimmunocytes in peritumoral inflammatory stroma has been shown to sustainthe complex microenvironment needed for lymphoid and solid tumormaintenance. L. Soucek, et al., Neoplasia 2011, 13, 1093-100; S.Ponader, et al., Blood 2012, 119, 1182-89; M. F. de Rooij, et al., Blood2012, 119, 2590-94. Taken together, these findings have suggested thatinhibition of BTK may offer an attractive strategy for treating B-cellneoplasms, other hematologic malignancies, and solid tumors.

Ibrutinib (PCI-32765, IMBRUVICA), is a first-in-class therapeutic BTKinhibitor. This orally delivered, small-molecule drug is being developedby Pharmacyclics, Inc. for the therapy of B-cell malignancies. Asdescribed above, in patients with heavily pretreated indolentnon-Hodgkin lymphoma (iNHL), mantle cell lymphoma (MCL), and CLL,ibrutinib showed substantial antitumor activity, inducing durableregressions of lymphadenopathy and splenomegaly in the majority ofpatients. R. H. Advani, et al., J. Clin. Oncol. 31, 88-94 (2013); J. C.Byrd, et al., N. Engl. J. Med. 2013, 369, 32-42; M. L. Wang, et al., N.Engl. J. Med. 2013, 369, 507-16. S. O'Brien, et al., Blood 2012, 119,1182-89. The pattern of changes in CLL was notable Inhibition of BTKwith ibrutinib caused rapid and substantial mobilization of malignantCLL cells from tissues sites into the peripheral blood, as described inJ. A. Woyach, et al., Blood 2014, 123, 1810-17; this effect wasconsistent with decreased adherence of CLL to protective stromal cells.S. Ponader, et al., Blood 2012, 119, 1182-89; M. F. de Rooij, et al.,Blood 2012, 119, 2590-94. Ibrutinib has been generally well tolerated.At dose levels associated with total BTK occupancy, not dose-limitingtoxicities were identified and subjects found the drug tolerable overperiods extending to >2.5 years.

Given the homology between BTK and interleukin-2 inducible tyrosinekinase (ITK), it has been recently confirmed that ibrutinib irreversiblybinds ITK. J. A. Dubovsky, et al., Blood 2013, 122, 2539-2549. ITKexpression in Fc receptor (FcR)-stimulated NK cells leads to increasedcalcium mobilization, granule release, and cytotoxicity. D. Khurana, etal., J. Immunol. 2007, 178, 3575-3582. As rituximab is a backbone oflymphoma therapy, with mechanisms of action including ADCC, as well asdirect induction of apoptosis and complement-dependent cytotoxicity andFcR stimulation is requisite for ADCC, we investigated if ibrutinib orFormula (II) (lacking ITK inhibition) influenced rituximab'santi-lymphoma activity in vitro by assessing NK cell IFN-γ secretion,degranulation by CD107a mobilization, and cytotoxicity by chromiumrelease using CD20⁺ cell lines and autologous patient samples withchronic lymphocytic leukemia (CLL).

Formula (II) is a more selective inhibitor than ibrutinib, as shownpreviously. Formula (II) is not a potent inhibitor of Itk kinase incontrast to ibrutinib (see Table 1). Itk kinase is required forFcR-stimulated NK cell function including calcium mobilization, granulerelease, and overall ADCC. As anti-CD20 antibodies like rituximab arestandard of care drugs, often as part of combination regimens, for thetreatment of CD20+B-cell malignancies, the potential of ibrutinib orFormula (II) to antagonize ADCC was evaluated in vitro. We hypothesizedthat Btk inhibitor, Formula (II) which does not have activity againstItk, may preserve NK cell function and therefore synergize rather thanantagonize rituximab-mediated ADCC. Rituximab-dependent NK-cell mediatedcytotoxicity was assessed using lymphoma cell lines as well asautologous CLL tumor cells.

Cell culture conditions were as follows. Cell lines Raji and DHL-4 weremaintained in RPMI 1630 supplemented with fetal bovine serum,L-glutamine, 2-mercaptoethanol and penicillin-streptomycin at 37° C. ina humidified incubator. The HER18 cells were maintained in DEMsupplemented with fetal bovine serum, penicillin-streptomycin and. Priorto assay, HER18 cells were harvested using trypsin-EDTA, washed withphosphate-buffered saline (PBS) containing 5% serum and viable cellswere counted. For culture of primary target cells, peripheral blood fromCLL patients was subject to density centrifugation to obtain peripheralblood mononuclear cells (PBMC). Cell preparations were washed and thensubject to positive selection of CD5⁺ CD19⁺ CLL cells using magneticbeads (MACS, Miltenyi Biotech). Cell preparations were used fresh afterselection. NK cells from CLL patients and healthy volunteers wereenriched from peripheral blood collected in sodium citrateanti-coagulant tubes and then subject to density centrifugation. Removalof non NK cells was performed using negative selection by MACSseparation. Freshly isolated NK cells were washed three times,enumerated, and then used immediately for ADCC assays.

Cytokine secretion was determined as follows. Rituximab andtrastuzumab-dependent NK-cell mediated degranulation and cytokinerelease were assessed using lymphoma and HER2+ breast cancer cell lines(DHL-4 and HER18, respectively). Target cells were cultured inflat-bottom plates containing 10 μg/mL of rituximab (DHL-4) ortrastuzumab (HER18) and test articles (0.1 or 1 μM ibrutinib, 1 μMFormula (II), or DMSO vehicle control). NK cells from healthy donorswere enriched as described above and then added to the target cells andincubated for 4 hours at 37° C. Triplicate cultures were performed on NKcells from donors. After incubation, supernatants were harvested,centrifuged briefly, and then analyzed for interferon-γ using anenzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis,Minn., USA).

Lytic granule release was determined as follows. NK cells from healthydonors were enriched and cultured in the presence of target cells,monoclonal antibodies and test articles as described above. After 4hours, the cultures were harvested and cells were pelleted, washed, andthen stained for flow cytometry evaluation. Degranulation was evaluatedvia by flow cytometery by externalization of CD107a, a protein normallypresent on the inner leaflet of lytic granules, and gating on NK cells(CD3-CD16⁺ lymphocytes). The percentage of CD107a positive NK cells wasquantified by comparison with a negative control (isotype control,unstained cells/FMO). Control cultures (NK cells cultured without targetcells, or NK, target cell co-cultures in the absence of appropriatemonoclonal antibody) were also evaluated; all experiments were performedin triplicate.

ADCC assays were performed as follows. Briefly, target cells (Raji orprimary CLL) were labeled by incubation at 37° C. with 100 μCi ⁵¹Cr for4 hours prior to co-culture with NK cells. Cells were washed,enumerated, and then added in triplicate to prepared 96-well platescontaining treated NK cells at an effector:target (E:T) ratio of 25:1.Rituximab (Genentech) was added to ADCC wells at concentrations of 0.1,1.0 or 10 μg/mL and the assays were briefly mixed and then centrifugedto collect cells at the bottom of the wells. The effect of NK cellnatural cytotoxicity was assessed in wells containing no rituximab.Cultures were incubated at 37° C. for 4 hours, and then centrifuged.Supernatants were harvested and ⁵¹Cr release was measured by liquidscintillation counting. All experiments were performed in triplicate.

Ibrutinib-inhibited rituximab-induced NK cell cytokine secretion in adose-dependent manner (0.1 and 1 μM) (FIG. 21: 48% p=0.018; 72% p=0.002,respectively). At 1 μM, Formula (II) did not significantly inhibitcytokine secretion (FIG. 21: 3.5%). Similarly, Formula (II) had noinhibitory effect on rituximab-stimulated NK cell degranulation (<2%)while ibrutinib reduced degranulation by ˜50% (p=0.24, FIG. 22). Formula(II) had no inhibitory effect while ibrutinib preventedtrastuzumab-stimulated NK cell cytokine release and degranulation by˜92% and ˜84% at 1 μM, respectively (FIG. 21 and FIG. 22: ***p=0.004,**p=0.002).

In Raji cells samples, ex vivo NK cell activity against autologous tumorcells was not inhibited by addition of Formula (II) at 1 μM, andincreased cell lysis was observed with increasing concentrations ofrituximab at a constant E:T ratio (FIG. 23). In primary CLL samples, exvivo NK cell activity against autologous tumor cells was not inhibitedby addition of Formula (II) at 1 μM, and increased cell lysis wasobserved with increasing concentrations of rituximab at a constant E:Tratio (FIG. 24). In contrast, addition of 1 μM ibrutinib completelyinhibited ADCC, with less than 10% cell lysis at any rituximabconcentration and no increase in cell lysis in the presence ofrituximab, compared with cultures without rituximab. The differencebetween Formula (II) and ibrutinib was highly significant in this assay(p=0.001). A plot highlighting the differences between Formula (II) andibrutinib at 10 μM is shown in FIG. 25.

In ADCC assays using healthy donor NK cells, antibody-dependent lysis ofrituximab-coated Raji cells was not inhibited by addition of 1 μMFormula (II) (FIG. 26). In these experiments, addition of rituximabstimulated a 5- to 8-fold increase in cell lysis at 0.1 and 1 μg/mL,compared with low (<20%) natural cytotoxicity in the absence ofrituximab. As previously reported, addition of 1 μM ibrutinib stronglyinhibited the antibody-dependent lysis of target cells, with less than20% cell lysis at all rituximab concentrations and no increase in ADCCwith at higher rituximab concentrations. The difference between Formula(II) and ibrutinib was highly significant in this assay (p=0.001).

Ibrutinib is clinically effective as monotherapy and in combination withrituximab, despite inhibition of ADCC in vitro and in vivo murine modelsdue to ibrutinib's secondary irreversible binding to ITK. Preclinically,the efficacy of therapeutics which do not inhibit NK cell function,including Formula (II), is superior to ibrutinib. Clinical investigationis needed to determine the impact of this finding on patients receivingrituximab as these results provide support for the unexpected propertyof Formula (II) as a better agent than ibrutinib to use in combinationwith antibodies that have ADCC as a mechanism of action.

Example 5 Effects of BTK Inhibition on Generalized NK Cell MediatedCytotoxicity

An assay was performed to assess the effects of BTK inhibition usingFormula (II) on generalized NK killing (non-ADCC killing). The targets(K562 cells) do not express MHC class I, so they do not inactivate NKcells. Target cells were grown to mid-log phase, and 5×10⁵ cells werelabeled in 100 μL of assay medium (IMDM with 10% FCS andpenicillin/streptomycin) with 100 μCi ⁵¹Cr for 1 hour at 37° C. Cellswere washed twice and resuspended in assay medium. A total of 5000target cells/well was used in the assay. Effector cells were resuspendedin assay medium, distributed on a V-bottom 96-well plate, and mixed withlabeled target cells at 40:1 E:T ratios. Maximum release was determinedby incubating target cells in 1% Triton X-100. For spontaneous release,targets were incubated without effectors in assay medium alone. After a1 minute centrifugation at 1000 rpm, plates were incubated for 4 and 16hours at 37° C. Supernatant was harvested and ⁵¹Cr release was measuredin a gamma counter. Percentage of specific release was calculated as(experimental release-spontaneous release)/(maximum release-spontaneousrelease)×100. The results are shown in FIG. 26.

Example 6 Effects of BTK Inhibition on T Cells

An assay was performed to assess the effects of BTK inhibition usingFormula (II) on T cells. Enriched CD4⁺ T cells are plated on 24-wellculture dishes that have been precoated 2 hr with 250 μL anti-TCRβ (0.5μg/mL) plus anti-CD28 (5 μg/mL) at 37° C. in PBS. The cells are thensupplemented with media containing BTK inhibitors along with the skewingcytokines as indicated in the following. The Th17 and Treg cultures aregrown for 4 days before analysis. The cells are maintained for anadditional 3 days with skewing cytokines (Th17; 20 ng/mL IL-6, 0.5 ng/mLTGF-β, 5 μg/mL IL-4, 5 μg/mL IFN-γ and Treg; 0.5 ng/mL TGF-β, 5 μg/mLIL-4, 5 μg/mL IFN-γ) and are supplemented with IL2 as a growth factor.

The results are shown in FIG. 27 and FIG. 28, and further illustrate thesurprising properties of Formula (II) in comparison to ibrutinib.Because of the lack of activity of Formula (II) on Itk and Txk, noadverse effects on Th17 and Treg development was observed. Sinceibrutinib inhibits both Itk and Txk, a profound inhibition of Th17 cellsand an increase in Treg development is observed, which is comparable tothe murine Itk/Txk double knock-out cells which were used as a control.

1. A method of treating chronic lymphocytic leukemia (CLL) or smalllymphocytic leukemia (SLL) comprising the step of orally administering,to a human in need thereof, the Bruton's tyrosine kinase (BTK)inhibitor, wherein the BTK inhibitor is a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, or hydrate thereof. 2.The method of claim 1, wherein the BTK inhibitor is administered oncedaily at a dose selected from the group consisting of 100 mg, 175 mg,250 mg, and 400 mg.
 3. The method of claim 1, wherein the BTK inhibitoris administered twice daily at a dose of 100 mg.
 4. The method of claim1, wherein the CLL increases monocytes and NK cells in peripheral bloodafter treatment with Formula (II) for a period selected from the groupconsisting of about 14 days, about 28 days, and about 56 days.
 5. Themethod of claim 1, wherein the CLL is selected from the group consistingof IgV_(H) mutation negative CLL, ZAP-70 positive CLL, ZAP-70 methylatedat CpG3 CLL, CD38 positive CLL, CLL with a 17p13.1 (17p) deletion, CLLwith a 11q22.3 (11q) deletion, CLL in a human sensitive toplatelet-mediated thrombosis, CLL in a human presently suffering fromplatelet-mediated thrombosis, CLL in a human previously suffering fromplatelet-mediated thrombosis, and combinations thereof.
 6. The method ofclaim 1, further comprising the step of administering a therapeuticallyeffective dose of an anti-CD20 antibody selected from the groupconsisting of rituximab, obinutuzumab, ofatumumab, veltuzumab,tositumomab, ibritumomab, and fragments, derivatives, conjugates,variants, radioisotope-labeled complexes, and biosimilars thereof. 7.The method of claim 1, further comprising the step of administering atherapeutically effective dose of an anticoagulant or antiplateletactive pharmaceutical ingredient.
 8. The method of claim 7, wherein theanticoagulant or antiplatelet active pharmaceutical ingredient isselected from the group consisting of acenocoumarol, anagrelide,anagrelide hydrochloride, abciximab, aloxiprin, antithrombin, apixaban,argatroban, aspirin, aspirin with extended-release dipyridamole,beraprost, betrixaban, bivalirudin, carbasalate calcium, cilostazol,clopidogrel, clopidogrel bisulfate, cloricromen, dabigatran etexilate,darexaban, dalteparin, dalteparin sodium, defibrotide, dicumarol,diphenadione, dipyridamole, ditazole, desirudin, edoxaban, enoxaparin,enoxaparin sodium, eptifibatide, fondaparinux, fondaparinux sodium,heparin, heparin sodium, heparin calcium, idraparinux, idraparinuxsodium, iloprost, indobufen, lepirudin, low molecular weight heparin,melagatran, nadroparin, otamixaban, parnaparin, phenindione,phenprocoumon, prasugrel, picotamide, prostacyclin, ramatroban,reviparin, rivaroxaban, sulodexide, terutroban, terutroban sodium,ticagrelor, ticlopidine, ticlopidine hydrochloride, tinzaparin,tinzaparin sodium, tirofiban, tirofiban hydrochloride, treprostinil,treprostinil sodium, triflusal, vorapaxar, warfarin, warfarin sodium,ximelagatran, salts thereof, solvates thereof, hydrates thereof, andcombinations thereof.
 9. A method of treating a hematological malignancyin a human comprising the step of administering a therapeuticallyeffective dose of a BTK inhibitor, wherein the BTK inhibitor is acompound of Formula (II):

or a pharmaceutically-acceptable salt, hydrate, or solvate thereof, andwherein the hematological malignancy is selected from the groupconsisting of non-Hodgkin's lymphoma (NHL), diffuse large B celllymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL),Hodgkin's lymphoma, B cell acute lymphoblastic leukemia (B-ALL),Burkitt's lymphoma, Waldenström's macroglobulinemia (WM), Burkitt'slymphoma, multiple myeloma, and myelofibrosis
 10. The method of claim 9,wherein the BTK inhibitor is administered once daily at a dose selectedfrom the group consisting of 100 mg, 175 mg, 250 mg, and 400 mg.
 11. Themethod of claim 9, wherein the BTK inhibitor is administered twice dailyat a dose of 100 mg.
 12. The method of claim 9, wherein thehematological malignancy increases monocytes and NK cells in peripheralblood after treatment with Formula (II) for a period selected from thegroup consisting of about 14 days, about 28 days, and about 56 days. 13.The method of claim 9, wherein the NHL is selected from the groupconsisting of indolent NHL and aggressive NHL.
 14. The method of claim9, wherein the DLBCL is selected from the group consisting of activatedB-cell like diffuse large B-cell lymphoma (DLBCL-ABC) and germinalcenter B-cell like diffuse large B-cell lymphoma (DLBCL-GCB).
 15. Themethod of claim 9, wherein the MCL is selected from the group consistingof mantle zone MCL, nodular MCL, diffuse MCL, and blastoid MCL.
 16. Themethod of claim 9, wherein the B-ALL is selected from the groupconsisting of early pre-B cell B-ALL, pre-B cell B-ALL, and mature Bcell B-ALL.
 17. The method of claim 9, wherein the Burkitt's lymphoma isselected from the group consisting of sporadic Burkitt's lymphoma,endemic Burkitt's lymphoma, and human immunodeficiency virus-associatedBurkitt's lymphoma.
 18. The method of claim 9, wherein the multiplemyeloma is selected from the group consisting of hyperdiploid multiplemyeloma and non-hyperdiploid multiple myeloma.
 19. The method of claim9, wherein the myelofibrosis is selected from the group consisting ofprimary myelofibrosis, myelofibrosis secondary to polycythemia vera, andmyelofibrosis secondary to essential thrombocythaemia.
 20. The method ofclaim 9, further comprising the step of administering a therapeuticallyeffective dose of an anti-CD20 antibody selected from the groupconsisting of rituximab, obinutuzumab, ofatumumab, veltuzumab,tositumomab, ibritumomab, and fragments, derivatives, conjugates,variants, radioisotope-labeled complexes, and biosimilars thereof. 21.The method of claim 9, further comprising the step of administering atherapeutically effective dose of an anticoagulant or antiplateletactive pharmaceutical ingredient.
 22. The method of claim 21, whereinthe anticoagulant or antiplatelet active pharmaceutical ingredient isselected from the group consisting of acenocoumarol, anagrelide,anagrelide hydrochloride, abciximab, aloxiprin, antithrombin, apixaban,argatroban, aspirin, aspirin with extended-release dipyridamole,beraprost, betrixaban, bivalirudin, carbasalate calcium, cilostazol,clopidogrel, clopidogrel bisulfate, cloricromen, dabigatran etexilate,darexaban, dalteparin, dalteparin sodium, defibrotide, dicumarol,diphenadione, dipyridamole, ditazole, desirudin, edoxaban, enoxaparin,enoxaparin sodium, eptifibatide, fondaparinux, fondaparinux sodium,heparin, heparin sodium, heparin calcium, idraparinux, idraparinuxsodium, iloprost, indobufen, lepirudin, low molecular weight heparin,melagatran, nadroparin, otamixaban, parnaparin, phenindione,phenprocoumon, prasugrel, picotamide, prostacyclin, ramatroban,reviparin, rivaroxaban, sulodexide, terutroban, terutroban sodium,ticagrelor, ticlopidine, ticlopidine hydrochloride, tinzaparin,tinzaparin sodium, tirofiban, tirofiban hydrochloride, treprostinil,treprostinil sodium, triflusal, vorapaxar, warfarin, warfarin sodium,ximelagatran, salts thereof, solvates thereof, hydrates thereof, andcombinations thereof.